HAHB11 Provides Improved Plant Yield and Tolerance to Abiotic Stress

ABSTRACT

The present invention provides isolated HaHB11 polypeptides and nucleic acids encoding the same. Also provided are methods of introducing a nucleic acid encoding HaHB11 polypeptides into a plant cell, plant part or plant, e.g., to increase tolerance to abiotic stress, to delay development and/or prolong the life span of a plant, and/or to increase yield from the plant. The invention also provides nucleic acids comprising HaHB11 promoter sequences and methods for expressing a nucleotide sequence of interest operably associated with the HaHB11 promoters of the invention in a plant cell, plant part, or plant. Also provided are transformed plants, plant tissues, plant cells and plant seed comprising the nucleic acids, expression cassettes and vectors of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/376,411, filed Jul. 20, 2015, which was the U.S. National Stage under35 C.F.R. § 371 of International Application No. PCT/US2013/024473,filed Feb. 1, 2013, which claims priority to and benefit of U.S.Provisional Application No. 61/594,133, filed Feb. 2, 2012. Thedisclosures of these prior applications are incorporated in theirentirety herein by reference thereto.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3181.0040002_Sequence_listing_ST25.txt; Size: 21,765 bytes, and Date ofCreation: Jan. 3, 2018) filed with the application is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to methods of expressing nucleic acids in plants.

BACKGROUND OF THE INVENTION

Global climate change influences the frequency and magnitude ofhydrological fluctuations, causing devastating events such as floods anddrought. These events, generating stress to plants, are the primarycause of crop loss worldwide, causing average yield losses of more than50% for major crops. Drought is the major factor that globally limitscrop productivity, and flooding represents another severe constraintthat limits crop growth and productivity. Both high and low extremes inprecipitation increasingly limit food, fiber and forest productionworldwide (Easterling et al., PNAS. 104:19679 (2007)).

Progress has been made in plant transformation for enhanced abioticstress tolerance through manipulation of either transcription and/orsignaling factors during the last two decades. However, none of thesetransformed crops has yet been marketed.

Transcription factors (TFs) regulate gene expression in response toenvironmental stresses. Bioinformatics studies have indicated thatArabidopsis has around 2000 genes encoding TFs (Riechmann et al.,Science 290:2105-2110 (2000); and Mitsuda et al., Plant and Cell Phys.50:1232-1248 (2009)), which are classified into different familiesaccording to the structure of their binding domains as follows: NAC,DOF, WRKY, bZIP, ERF/AP2, MYB, Zn-finger and homeodomain (HD). HD is a60 amino acid DNA-binding domain that is present in TFs across alleukaryotic organisms, and which is encoded by a 180 bp sequencedesignated as Homeobox (HB). In plants, HD proteins are divided intodifferent families, for example, HD-Zip, PHD finger, Bell, ZF-HD, WOXand KNOX (Chan et al., Biochim. Biophys. Acta 1442:1-19 (1998); Ariel etal., Trends Plant Sci. 12:419-426 (2007)). Members of the HD-Zip familyexhibit an association of a HD and a downstream leucine zipper motif(LZ). This association in a single protein is unique to plants (Schenaet al., PNAS 89, 3894-3898 (1992)). HD-Zip proteins can be divided intofour subfamilies, named I to IV, according to several structural andfunctional features.

SUMMARY OF THE INVENTION

The invention encompasses HaHB11 related compositions and their use.HaHB11.1 encodes a 175 amino acid protein, initially identified inHelianthus annuus, belonging to the HD-Zip family of transcriptionfactors. HaHB11.2 encodes a 181 amino acid protein, initially identifiedin Helianthus annuus, belonging to the HD-Zip family of transcriptionfactors. Recently, phylogenetic trees constructed with proteins fromseveral species resolved HD-Zip I members in six groups according to theconservation within and outside of the HD-Zip domain (Arce et al., BMCPlant Biol. 11:42 (2011)). Neither HaHB11 nor the well-characterizedHaHB4 transcription factor (Gago et al., Plant Cell Environ. 25:633-640(2002); Dezar et al., Transgenic Res. 14:429-440 (2005), Dezar et al.,Plant Sci. 169:447-459 (2005); Manavella et al., Plant J. 48:125-137(2006); Cabello et al., 2007; Manavella et al., Plant Physiol. Biochem.46:860-867 (2008), Manavella et al., Plant J. 56:376-388 (2008), andManavella et al., J. Exp. Bot. 59: 3143-3155 (2008)) were resolved intoany of these six groupings, indicating that they are divergent membersand probably possess differential functions (Arce et al., BMC PlantBiol. 11:42 (2011)).

The inventors have found that HaHB11 provides increased tolerance toabiotic stress and has dual functionality, participating in both droughtand flooding tolerances. The inventors have also found that HaHB11provides increased salinity tolerance. Transgenic plants expressingHaHB11 exhibit longer roots. Moreover, transgenic plants expressingHaHB11 exhibit larger rosettes and improved yield as compared withcontrols under normal growth conditions (e.g., normal irrigation and nosalt stress) and conditions of abiotic stress.

Accordingly, as a first aspect, the invention provides a transgenicplant stably transformed with an isolated nucleic acid encoding apolypeptide selected from the group consisting of:

(a) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 111 to 175 of SEQ IDNO:3; and

(b) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence that is atleast about 70% similar to amino acids 111 to 175 of SEQ ID NO:3.

In some embodiments, the invention provides a transgenic plant stablytransformed with an isolated nucleic acid encoding a polypeptideselected from the group consisting of:

(a) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 114 to 181 of SEQ IDNO:10; and

(b) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence that is atleast about 96% similar or at least 96%, 97%, 98% or 99% identical toamino acids 114 to 181 of SEQ ID NO:10.

In some embodiments, the invention provides a transgenic plant stablytransformed with an isolated nucleic acid encoding a polypeptideselected from the group consisting of:

(a) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 115 to 113 of SEQ IDNO:10; and

(b) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence that is atleast about at least 96%, 97%, 98% or 99% identical to amino acids 15 to181 of SEQ ID NO:10.

In some embodiments, the invention provides a transgenic plant stablytransformed with an isolated nucleic acid encoding a polypeptideselected from the group consisting of:

(a) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 114 to 180 of SEQ IDNO:11, amino acids 111 to 179 of SEQ ID NO:12; amino acids 111 to 177 ofSEQ ID NO:13, amino acids 113 to 183 of SEQ ID NO:14 or amino acids 112to 186 of SEQ ID NO:15;

(b) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence that is atleast about 96% similar or at least 96%, 97%, 98% or 99% identical toamino acids amino acids 114 to 180 of SEQ ID NO:11, amino acids 111 to179 of SEQ ID NO:12; amino acids 111 to 177 of SEQ ID NO:13, amino acids113 to 183 of SEQ ID NO:14, or amino acids 112 to 186 of SEQ ID NO:15.

In representative embodiments, the transgenic plant has an increasedtolerance to abiotic stress and/or increased yield.

As a further aspect, the invention provides a transgenic plant stablytransformed with an isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO:2;

(b) a nucleotide sequence comprising at least 100 consecutivenucleotides of the nucleotide sequence of SEQ ID NO:2;

(c) a nucleotide sequence having at least 95% sequence identity to thenucleotide sequence of (a) or (b);

(d) a nucleotide sequence that hybridizes to the complete complement ofthe nucleotide sequence of (a) or (b) under stringent hybridizationconditions; and

(e) a nucleotide sequence that differs from the nucleotide sequence ofany of (a) to (d) due to the degeneracy of the genetic code.

In some embodiments, the invention provides a transgenic plant stablytransformed with an isolated nucleic acid comprising a nucleotidesequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO:9;

(b) a nucleotide sequence having at least 96% sequence identity to thenucleotide sequence of (a); and

(c) a nucleotide sequence that differs from the nucleotide sequence ofany of (a) or (b) due to the degeneracy of the genetic code.

In representative embodiments, the transgenic plant has an increasedtolerance to abiotic stress and/or increased yield.

As yet another aspect, the invention provides a method of increasingyield and/or increasing tolerance of a plant to abiotic stress, themethod comprising:

(a) stably transforming a plant cell with an isolated nucleic acidencoding a polypeptide selected from the group consisting of:

(i) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (b) amino acids 111 to 175 of SEQ IDNO:3; and

(ii) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7 and (b) an amino acid sequence that is atleast about 70% similar to amino acids 111 to 175 of SEQ ID NO:3.

In some embodiments, the invention provides a method of increasing yieldand/or increasing tolerance of a plant to abiotic stress, the methodcomprising:

(a) stably transforming a plant cell with an isolated nucleic acidencoding a polypeptide selected from the group consisting of:

(i) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (b) amino acids 114-181 of SEQ ID NO:10;and

(ii) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (b) an amino acid sequence that is atleast about that is at least about 96% similar or at least 96%, 97%, 98%or 99% identical to amino acids 114 to 180 of SEQ ID NO:10.

In some embodiments, the invention provides a method of increasing yieldand/or increasing tolerance of a plant to abiotic stress, the methodcomprising:

(a) stably transforming a plant cell with an isolated nucleic acidencoding a polypeptide selected from the group consisting of:

(i) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (b) amino acids 114 to 180 of SEQ IDNO:11, amino acids 111 to 179 of SEQ ID NO:12; amino acids 111 to 177 ofSEQ ID NO:13, amino acids 113 to 183 of SEQ ID NO:14, or amino acids 112to 186 of SEQ ID NO:15; and

(ii) a polypeptide comprising (a) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (b) an amino acid sequence that is atleast about that is at least about 96% similar or at least 96%, 97%, 98%or 99% identical to amino acids 114 to 180 of SEQ ID NO:11, amino acids111 to 179 of SEQ ID NO:12; amino acids 111 to 177 of SEQ ID NO:13,amino acids 113 to 183 of SEQ ID NO:14, or amino acids 112 to 186 of SEQID NO:15.

Still further, the invention provides a method of increasing yieldand/or increasing tolerance of a plant to abiotic stress, the methodcomprising:

(a) stably transforming a plant cell with an isolated nucleic acidcomprising a nucleotide sequence selected from the group consisting of:

(i) the nucleotide sequence of SEQ ID NO:2;

(ii) a nucleotide sequence comprising at least 100 consecutivenucleotides of the nucleotide sequence of SEQ ID NO:2;

(iii) a nucleotide sequence having at least 95% sequence identity to thenucleotide sequence of (i) or (ii);

(iv) a nucleotide sequence that hybridizes to the complete complement ofthe nucleotide sequence of (i) or (ii) under stringent hybridizationconditions; and

(v) a nucleotide sequence that differs from the nucleotide sequence ofany of (i) to (iv) due to the degeneracy of the genetic code.

(b) regenerating a stably transformed plant from the stably transformedplant cell of (a); and

(c) expressing the nucleotide sequence in the plant.

In additional embodiments, the invention provides a method of increasingyield and/or increasing tolerance of a plant to abiotic stress, themethod comprising:

(a) stably transforming a plant cell with an isolated nucleic acidcomprising a nucleotide sequence selected from the group consisting of:

the nucleotide sequence of SEQ ID NO:9;

(ii) a nucleotide sequence having at least 96% sequence identity to thenucleotide sequence of (i);

(iii) a nucleotide sequence that differs from the nucleotide sequence ofany of (i) to (ii) due to the degeneracy of the genetic code.

(b) regenerating a stably transformed plant from the stably transformedplant cell of (a); and

(c) expressing the nucleotide sequence in the plant.

As another aspect, the invention provides an isolated nucleic acidcomprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6;

(b) a nucleotide sequence comprising at least 100 consecutivenucleotides of the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:16;

(c) a nucleotide sequence that hybridizes to the complete complement ofthe nucleotide sequence of (a) or (b) under stringent hybridizationconditions; and

(d) a nucleotide sequence having at least 95% sequence identity to thenucleotide sequences of any of (a) to (c).

In another embodiment, the invention provides an isolated nucleic acidcomprising a nucleotide sequence selected from the group consisting of:

(a) the nucleotide sequence of SEQ ID NO:16;

(b) a nucleotide sequence comprising at least 25 consecutive nucleotidesof the nucleotide sequence of nucleotides 1 to 800 of SEQ ID NO:16;

(c) a nucleotide sequence that hybridizes to the complete complement ofnucleotides 1-200, 200-400, 400-600 and/or 600-800 of SEQ ID NO:16 understringent hybridization conditions; and

(d) a nucleotide sequence having at least 95% sequence identity to thenucleotide sequences of any of (a) to (c).

In another embodiment, the invention provides an isolated nucleic acidcomprising a nucleotide sequence selected from the group consisting of:

-   -   (a) the nucleotide sequence of SEQ ID NO:16;    -   (b) a nucleotide sequence comprising at least 100 consecutive        nucleotides of nucleotides 1 to 800 of SEQ ID NO:16;    -   (c) a nucleotide sequence that hybridizes to the complete        complement of the nucleotide sequence of (b) under stringent        hybridization conditions; and    -   (d) a nucleotide sequence having at least 95% sequence identity        to the nucleotide sequences of any of (a) to (c). In some        embodiments, the isolated nucleic acid comprises the nucleotide        sequence of SEQ ID NO:9.

Also provided are expression cassettes, vectors, cells, plants and plantparts comprising the isolated nucleic acids of the invention operablyassociated with a nucleotide sequence of interest.

In some embodiments, the invention encompasses and expression cassettecomprising an isolated HaHB11 nucleic acid operably associated with anucleotide sequence of interest. In further embodiments, the nucleotidesequence of interest is selected from the group consisting of: (a) thenucleotide sequence of SEQ ID NO:2; (b) the nucleotide sequence of SEQID NO:9; (c) a nucleotide sequence comprising at least 100 consecutivenucleotides of the nucleotide sequence of (a) or (b); (d) a nucleotidesequence having at least 95% sequence identity to the nucleotidesequence of (a) or (b); (e) a nucleotide sequence that hybridizes to thecomplete complement of the nucleotide sequence of (a) or (b) understringent hybridization conditions; and (f) a nucleotide sequence thatdiffers from the nucleotide sequence of any of (a) to (e) due to thedegeneracy of the genetic code. In additional embodiments, thenucleotide sequence of interest encodes a polypeptide selected from thegroup consisting of: (a) a HAHB11 polypeptide of SEQ ID NO:3; (b) aHAHB11 polypeptide of SEQ ID NO:10; (c) a polypeptide comprising atleast 100 consecutive amino acids of (a) or (b); and (d) a polypeptidesequence having at least 95% sequence identity to the sequence of (a) or(b). In some embodiments the isolated nucleic acid is operablyassociated with a heterologous nucleotide sequence of interest. In otherembodiments, the expression cassette contains a sequence that encodes aselectable marker. Cells, plants and plant parts transformed with thesenucleic acids, vectors, and expression cassettes are also encompassed bythe invention. In particular embodiments, the transformed plants aremonocots. In other embodiments, the transformed plants are dicots. Inparticular embodiments, the cells, plants and/or plant parts correspondto sunflower, wheat, maize, soybean, rice, alfalfa or Arabidopsis.

As an additional aspect, the invention also encompasses productsharvested from the plants of the invention and processed productsproduced therefrom.

The invention also provides seed produced from the plants of theinvention and seed comprising the isolated nucleic acids and expressioncassettes of the invention.

As still a further aspect, the invention provides a method ofintroducing a nucleic acid into a plant, plant part or plant cell, themethod comprising transforming the plant, plant part or plant cell withan isolated nucleic acid, expression cassette or vector of theinvention.

As yet another aspect, the invention provides a method of stablyexpressing a nucleotide sequence of interest in a plant, the methodcomprising:

(a) stably transforming a plant cell with an expression cassette orvector of the invention;

(b) regenerating a stably transformed plant from the stably transformedplant cell of (a); and

(c) expressing the nucleotide sequence of interest in the plant.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the sunflower HaHB11 gene. Thepromoter region is shown as crosshatched, and the coding region asstippled. Putative cis acting elements are signaled in the promoter aswell as the conserved HD Zip domain in the coding sequence.

FIG. 1B is a schematic representation of the sunflower HaHB11 protein(SEQ ID NO:13). The homeodomain (HD) is shown in bold. The leucinezipper (Zip) is underlined; each leucine (L) is also shown in bold. Theamino terminal domain is shown in italics. The carboxyl-terminus isshown in double underlining. Putative casein-phosphorylation sites areshown in black shadowing with white letters. A putative transactivationsite is shown in black shadowing with gray letters. A putative proteinkinase phosphorylation site is shown in gray letters.

FIG. 2A. Polynucleotide sequence alignment of HaHB11.1 (SEQ. ID NO:2)and HaHB11.2 (SEQ ID NO:9) sequence variants.

FIG. 2B. Protein sequence alignment of HaHB11.1 (SEQ. ID NO:3) andHaHB11.2 (SEQ ID NO:10) sequence variants

FIG. 2C. Protein sequence alignment of Asteraceae family HaHB11orthologs/variants from Helianthus annuus (Hann; SEQ ID NO:3 and SEQ IDNO:10), Helianthus tuberosus, (Htub; SEQ ID NO:11, SEQ ID NO:14 and SEQID NO:15), Helianthus argophyllus (Harg; SEQ ID NO:12) and Helianthusciliaris (Hcil; SEQ ID NO:13).

FIG. 3: HaHB11 expression pattern in 7-day-old sunflower seedlings.HaHB11 transcript levels were quantified by qPCR and normalized withhousekeeping ACTIN transcripts. The values obtained were normalized withrespect to the value measured in the root sample, arbitrarily assigned avalue of one. Error bars correspond to SD among three biologicalreplicas.

FIG. 4: HaHB11 expression pattern in 21-day-old sunflower plants. HaHB11transcript levels were quantified by qPCR and normalized withhousekeeping ACTIN transcripts. The values obtained were normalized withrespect to the value measured in root sample, arbitrarily assigned avalue of one. Error bars correspond to SD among three biologicalreplicas.

FIG. 5: HaHB11 expression pattern in 7-day-old sunflower seedlings underdifferent treatments. HaHB11 transcript levels were quantified by qPCRand normalized with housekeeping ACTIN transcripts. The values obtainedwere normalized with respect to the value measured in root sample,arbitrarily assigned a value of one. Error bars correspond to SD amongthree biological replicas.

FIG. 6: HaHB11 expression pattern in leaf discs of 21-day-old sunflowerplants under different treatments. HaHB11 transcript levels werequantified by qPCR and normalized with housekeeping ACTIN transcripts.The obtained values were normalized with respect to the value measuredin root sample, arbitrarily assigned a value of one. Error barscorrespond to SD among three biological replicas.

FIG. 7: HaHB11 expression in front of drought and salinity stresses.Sunflower R2 plants were subjected to drought (left panel) or salinity(right panel) stresses as described in the Experimental section. In theright panel, 3d50 means 3 days after 50 mM NaCl addition. Leaf diskswere frozen at the times indicated in the figures and HaHB11 transcriptlevels were quantified by qPCR and normalized with housekeeping ACTINtranscripts. The obtained values were normalized with respect to thevalue measured in the control sample, arbitrarily assigned a value ofone. Error bars correspond to SD among three biological replicas.

FIG. 8: Relative expression levels of HaHB11 in Arabidopsis transgenicplants. RNA was extracted from each genotype (several plants) andtranscript levels quantified by qPCR. All the values were normalizedwith respect to the value measured in line 15, arbitrarily assigned avalue of one.

FIG. 9: Phenotype of transgenic plants ectopically expressing HaHB11compared to WT. Left: 25-day-old plants grown at normal conditions.Right: Number of rosette leaves in the transition from the vegetative tothe reproductive stage and an amplification of the photograph of thetransgenic leaves.

FIG. 10: Stem length measured during the life cycle of 35S:HaHB11 and WTplants. Stem length was measured starting at the transition phase andthereafter every 3-5 days until the end of the life cycle.

FIG. 11: Inflorescence morphology. Inflorescences of 35-day-old plantsgrown in standard conditions.

FIG. 12: Phenotype of plants grown in mannitol. Upper panel: plantsgrown on MS. Middle and lower panel: Plants grown in mannitol 100 mM.

FIG. 13: Hypocotyl and root length. Hypocotyl and root length of7-day-old plants grown on MS medium was measured. Average lengths andstandard deviations were calculated from n=40 seedlings.

FIG. 14: Transgenic plants are more tolerant to drought than their WTcontrols. Illustrative assay performed with 25-day-old 35S:HaHB11 and WTplants, water-starved during 15 days. The illustrative photograph wastaken during the assay. Right: a table showing the survival rate of eachgenotype. Statistical analysis was performed with 64 plants from eachgenotype.

FIG. 15: Water loss treatment under drought stress. Drought toleranceassay performed with 25-day-old 35S:HaHB11 and WT plants during 15 days.Water loss was measured at the times indicated in the figure. W1 is theweight of dehydrated leaves and W2 the weight of re-watered leaves.

FIG. 16: Stomatal closure under drought stress. 25-day-old 35S:HaHB11and WT plants grown on MS medium and then dehydrated on paper during 15minutes. Photographs were taken using an optical microscope. Theobservation was performed with n=50 stomata for each genotype.

FIG. 17: Stomatal closure during water stress treatment in plants grownon soil. 25-day-old 35S:HaHB11 and WT plants were subjected to droughtduring 15 days. Water loss during the treatment was quantified at thetimes indicated in the figure as described above. W1 is the weight ofdehydrated leaves and W2 the weight of re-watered leaves. Stomatalclosure was estimated by microscopic observation at specific pointsduring the assay.

FIG. 18: Effect of ABA 10 uM on stomata closure in WT and transgenicplants. 25-day-old 35S:HaHB11 and WT plants grown on MS medium weretreated with ABA 10 uM for 1 hour. Stomatal closure was quantifiedbefore and after ABA treatment in n=50 stomata.

FIG. 19: Water use efficiency during drought assay. 25-day-old35S:HaHB11 and WT plants grown on soil and dried during 14 days. Soilpots were weighed every day during the assay. Circles indicate theaverage day on which plant of each genotype died.

FIG. 20: Transgenic plants consumed less water than WT during a stresstreatment. 25-day-old 35S:HaHB11 and WT plants were grown on soil andwatering was stopped during 14 days. Soil pots were weighed every twodays during the assay and water was added to those that needed it tomaintain the same water volume in all the pots. In general, highexpressing HaHB11 transgenic plants did not need any addition of water.

FIG. 21A, FIG. 21B, and FIG. 21C: Transgenic plants bearing theconstruct 35S:HaHB11 exhibited a larger rosette and a significantlyhigher yield. FIG. 21A: 40-day-old WT and 35S:HaHB11 plants were grownon soil and normally irrigated during the whole life cycle. FIG. 21B:the same but with 50-day-old plant. FIG. 21C: Yield was calculated froman experiment performed with 12 plants of each genotype.

FIG. 22: Expression levels of selected genes involved in ABA synthesisand signaling in 35S:HaHB11 and WT plants. RNA from 21-day-old35S:HaHB11 and WT plants grown on MS medium treated with ABA 100 1-1Mduring 1 hour were isolated and quantified by qPCR. Statistical analysiswas performed from three biological triplicates.

FIG. 23: Expression levels of genes involved in abiotic stressprotection in 35S:HaHB11 and WT plants. RNA from 21-day-old 35S:HaHB11and WT plants grown on MS medium were treated with ABA 100 1-1M during 1hour were isolated and analyzed by qPCR. Statistics analysis wasperformed with three biological triplicates.

FIG. 24: Transgenic plants were tolerant to salinity stress. 21-day-old35S:HaHB11 and WT plants were watered with increasing NaClconcentrations up to 400 mM during a 21 day period. The photograph wastaken 7 days after the end of the assay. Statistics was performed withn=32 plants for each genotype.

FIG. 25: Histochemical GUS staining in PrH11:GUS plants. GUS detectionin 7-day-old PrH11:GUS plants grown on MS medium.

FIG. 26: Histochemical GUS staining in PrH11:GUS plants. GUS detectionin 14-day-old PrH11: GUS plants grown on MS medium.

FIG. 27A: 35S:HaHB11 plants tolerated submergence better than theircontrols. 21-day-old 35S:HaHB11 and WT (101) plants grown on soil andsubmerged during six days. The photograph was taken six days afterrecovery under standard conditions. FIG. 27B-chlorophyll content of thesame plants.

FIG. 27C. Water content quantified in plants subjected for six days to asubmergence treatment and then, desubmerged. The x axis indicates daysafter the end of the submergence treatment.

FIG. 28: Chlorophyll content after 6 days of waterlogging. 21-day-old35S:HaHB11 and WT plants grown on soil with their roots submerged forsix days. Chlorophyll content was measured after the treatment.

FIG. 29: Tolerance to waterlogging. 21-day-old 35S:HaHB11, and WT plantsgrown on soil with their roots submerged for six days. Photograph wastaken and percentage of survivors was calculated 6 days after recovery.

FIG. 30: Hypothetical model of HaHB11 function in plants.

FIG. 31: Weight of seeds (g/plant) was obtained after harvesting of35S:HaHB11 overexpressing and WT plants grown in under normal conditionsor subjected to a mild stress (3 days of waterlogging) as described inthe Experimental procedures. Seed weight obtained for HaHB11 and WTplants subjected to a moderate waterlogging stress. HaHB11 plantsexhibit approximately twice the yield of WT plants.

FIG. 32A depicts a qualitative assay for starch using a lugol stainingtechnique. FIG. 32B—presents a quantitation of the accumulated starchduring the day. Starch quantification was performed at the end of theday. As depicted, transgenic plants exhibited more starch than WT underthe tested conditions.

FIG. 33: Histochemical detection of GUS enzymatic activity in transgenicplants containing the long HaHB11 promoter (ProH11 long; SEQ ID NO:16)ProH11 long: GUS plants. 25-day-old plants were grown on MS medium atnormal conditions or in presence of the fitohormone ABA for 1.5 hours.The figure demonstrate that the location of the expression did notchange after ABA treatment. However, the intensity of the signalsignificantly increased in all the tissues, indicating an up-regulationof this promoter by ABA. Cotyledons (photographs A and F), hypocotyls(photographs B and G), petioles (photographs C and H) and roots(photographs D-J).

FIG. 34: Transgenic plants bearing the construct HaHB11 long promoterconstruct (SEQ IN NO:17) PromH11 long:HaHB11 exhibited significantlylonger roots compared to the WT plants (transgenic for 35S::GUS).

FIG. 35: depicts the results of a transcription activation assay inwhich Sacharomyces cerevisiae, strain Y187, was transformed withconstructs encoding HaHB11.1 (SEQ ID NO:3) and HaHB11.2 (SEQ ID NO:10)).The activation assay was performed as described in the methods. Theresults indicate that HaHB11 acts as an activator, at least in the yeastsystem, and that both HaHB11.1 (SEQ ID NO:3) and HaHB11.2 do not appearto differ significantly in this ability in the assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of newtranscription factor in Helianthus annuus, designated as HaHB11. HaHB11transgenic plants display longer roots than wild type plants (FIG. 34).Plants over-expressing HaHB11 exhibit increased tolerance to abioticstress (including drought, salinity, waterlogging, submergence anddesubmergence [post-water submergence] stress). In addition, HaHB11over-expressers are taller and demonstrate an increased yield of seedunder non-stressed conditions (e.g., normal cultivation conditions).

The present invention will now be described in more detail withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted. To illustrate, if thespecification states that a composition comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

I. Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “about,” as used herein when referring to a measurable valuesuch as a dosage or time period and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The terms “comprise,” “comprises” and “comprising” as used herein,specify the presence of the stated features, integers, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, integers, steps,operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461,463(CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus,the term “consisting essentially of” when used in a claim or thedescription of this invention is not intended to be interpreted to beequivalent to “comprising.”

An “abiotic stress” is a stressor from one or more outside, non-livingfactors that adversely affects the productivity and/or the survival ofthe organism. Abiotic stressors include, but are not limited to:drought, flooding stress (e.g., due to waterlogging and/or submergence),stress following the removal of a flooding stressor (e.g., dehydrationin the period following removal of a flooding stress such asdesubmergence stress), salt stress (e.g., high or excessive saltconditions), high winds, heat or high temperature, cold temperature,freezing, fire, high light intensity, low light intensity, ozone, poorpH (too alkaline and/or too acidic), soil compaction and/or highradiation. Those skilled in the art will appreciate that an abioticstress will depend on the preferred conditions for that organism, andmay well vary due to the presence of other biotic and/or abioticstressors.

Parameters for abiotic stress factors are species specific and evenvariety specific and therefore vary widely according to thespecies/variety exposed to the abiotic stress. Thus, while one speciesmay be severely impacted by a salinity level of 4.0 dS m⁻¹, anotherspecies may not be affected until at least a salinity level of 6.0 dSm⁻¹ or even 10.0 dS m⁻¹. See, for example, Blaylock, A. D. (“Soilsalinity, salt tolerance, and growth potential of horticultural andlandscape plants,” Univ. Wyoming, Cooperative Extension Service,Bulletin B-988, February 1994) in which different plants are categorizedas sensitive, moderately sensitive, moderately tolerant and tolerantdepending on the level of soil salinity required to affect plant growth.Thus, for example, the level of salinity that is excessive or high for asensitive plant species or variety is not the same level of salinitythat is excessive or high for a moderately sensitive plant or a tolerantplant. The same is true for other types of abiotic stress such asdrought, waterlogging and submergence stress. Thus, a level of droughtthat can be tolerated by a sensitive plant species/variety is differentfrom the level of drought that can be tolerated by a plantspecies/variety that is more drought tolerant. Likewise, the level offlooding (e.g., at the roots and/or the aerial parts of the plant) thatcan be tolerated by a sensitive plant species/variety is different fromthat for a plant that is more tolerant to excessive water (e.g., wetroots and/or submergence).

“Severe” abiotic stress results in death in control plants (e.g., plantsnot expressing an HaHB11 polypeptide of the invention), for example, atleast about 10%, 20%, 30%, 40%, 50% or even more control plants die,whereas the plants of the invention (e.g., expressing an HaHB11polypeptide of the invention) exhibit an increased survival, or even noplant death, and/or are less severely affected as compared withcontrols.

“Mild” abiotic stress is defined herein as conditions in which controlplants do not die but their production is very low (e.g., reduced by atleast about 30%, 40%, 50% or more), whereas the plants of the invention(e.g., expressing an HaHB1 polypeptide of the invention) exhibitincreased production as compared with and/or are less severely affected,as compared with controls. For example, mild drought stress can beachieved by providing about 50% of the water needed to achieve maximumyield.

“Normal” growth conditions are conditions in which the plants are notexposed to significant biotic, abiotic, toxicologic or nutritionalstress, e.g., conditions in which the plants are well irrigated andexposed to normal salt conditions. In particular embodiments, “normal”growth conditions are conditions in which plants are exposed to nodetectable (e.g., measurable) biotic, abiotic, toxicologic ornutritional stress.

Thus, an “increased tolerance to abiotic stress” (and similar terms) asused herein refers to the ability of a plant or part thereof exposed toabiotic stress and transformed with an isolated or recombinant nucleicacid of the invention (e.g., encoding an HaHB11 polypeptide of theinvention) to withstand a given abiotic stress better than a controlplant or part thereof (e.g., a plant or part thereof that has beenexposed to the same abiotic stress but has not been transformed with anisolated or recombinant nucleic acid molecule of the invention).Increased tolerance to abiotic stress can be measured using a variety ofparameters including, but not limited to, the size and/or number ofplants or parts thereof (e.g., leaf number and/or size), productivity oryield (e.g., of seed), relative water content, electrolyte leakage,stomata conductance, photosynthetic rate, internal CO₂ concentration,transpiration rate, chlorophyll fluorescence. Thus, in some embodimentsof the invention, a transformed plant or part thereof comprising anisolated or recombinant nucleic acid molecule of the invention, therebyhaving increased tolerance to the abiotic stress, would have, forexample, greater growth (e.g., plant height) and/or survival and/oryield as compared with a plant or part thereof exposed to the samestress but not having been transformed with an isolated or recombinantnucleic acid molecule of the invention.

As used herein, “flooding stress” includes any stress induced by excesswater and encompasses both waterlogging stress and submergence stress.

The term “waterlogging stress,” as used herein, includes the stressinduced by water covering the roots and soil, but the aerial portion ofthe plant is not necessarily covered by water. For example, waterloggingstress can be induced under conditions in which the soil surrounding theroots is saturated with water. In embodiments of the invention, lessthan about 50% or 25% of the aerial portion of the plant is submergedunder water. In embodiments of the invention, essentially none of theaerial portion of the plant is submerged (e.g., less than about 5% or10%).

The term “submergence stress,” as used herein, includes the stressinduced when the aerial portion of the plant is substantially underwater. In embodiments of the invention, at least about 15%, 20%, 25%,30%, 40%, 50%, 75%, 85%, 90% or 95% of the aerial portions of the plantare submerged under water. In embodiments of the invention, the entireaerial portion of the plant is submerged under water.

An “increased yield” (and similar terms) as used herein refers to anenhanced or elevated production of a commercially and/or agriculturallyimportant plant, plant biomass, plant part (e.g., roots, tubers, seed,leaves, fruit), plant material (e.g., an extract) and/or other productproduced by the plant (e.g., a recombinant polypeptide) by a plant orpart thereof exposed to abiotic stress and transformed with an isolatedor recombinant nucleic acid of the invention (e.g., encoding an HaHB11polypeptide of the invention) as compared with a control plant or partthereof (e.g., a plant or part thereof that has been exposed to the sameabiotic stress but has not been transformed with an isolated orrecombinant nucleic acid molecule of the invention).

The term “modulate” (and grammatical variations) refers to an increaseor decrease.

As used herein, the terms “increase,” “increases,” “increased,”“increasing” and similar terms indicate an elevation of at least about25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

As used herein, the terms “reduce,” “reduces,” “reduced,” “reduction”and similar terms mean a decrease of at least about 25%, 35%, 50%, 75%,80%, 85%, 90%, 95%, 97% or more. In particular embodiments, thereduction results in no or essentially no (i.e., an insignificantamount, e.g., less than about 10% or even 5%) detectable activity oramount.

As used herein, the term “heterologous” means foreign, exogenous,non-native and/or non-naturally occurring.

As used here, “homologous” means native. For example, a homologousnucleotide sequence or amino acid sequence is a nucleotide sequence oramino acid sequence naturally associated with a host cell into which itis introduced, a homologous promoter sequence is the promoter sequencethat is naturally associated with a coding sequence, and the like.

As used herein a “chimeric nucleic acid,” “chimeric nucleotide sequence”or “chimeric polynucleotide” comprises a promoter operably linked to anucleotide sequence of interest that is heterologous to the promoter (orvice versa). In particular embodiments, the “chimeric nucleic acid,”“chimeric nucleotide sequence” or “chimeric polynucleotide” comprises aHaHB11 promoter element operably associated with a heterologousnucleotide sequence of interest to be transcribed. In otherrepresentative embodiments, the “chimeric nucleic acid,” “chimericnucleotide sequence” or “chimeric polynucleotide” comprises a HaHB11coding sequence operably associated with a heterologous promoter.

A “promoter” is a nucleotide sequence that controls or regulates thetranscription of a nucleotide sequence (i.e., a coding sequence) that isoperatively associated with the promoter. The coding sequence may encodea polypeptide and/or a functional RNA Typically, a “promoter” refers toa nucleotide sequence that contains a binding site for RNA polymerase-IIand directs the initiation of transcription. In general, promoters arefound 5′, or upstream, relative to the start of the coding region of thecorresponding coding sequence. The promoter region may comprise otherelements that act as regulators of gene expression. These include a TATAbox consensus sequence, and often a CAAT box consensus sequence(Breathnach et al., Annu. Rev. Biochem. 50:349 (1981)). In plants, theCAAT box may be substituted by the AGGA box (Messing et al., (1983) inGenetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender(eds.), Plenum Press, pp. 211-227). The promoter region, including allthe ancillary regulatory elements, typically contain between about 100and 1000 nucleotides, but can be as long as 2 kb, 3 kb, 4 kb or longerin length. Promoters according to the present invention can function asconstitutive and/or inducible regulatory elements.

A “functional” RNA includes any untranslated RNA that has a biologicalfunction in a cell, e.g., regulation of gene expression. Such functionalRNAs include but are not limited to siRNA, shRNA, miRNA, antisense RNA,ribozymes, and the like.

By “operably linked” or “operably associated” as used herein, it ismeant that the indicated elements are functionally related to eachother, and are also generally physically related. For example, apromoter is operatively linked or operably associated to a codingsequence (e.g., nucleotide sequence of interest) if it controls thetranscription of the sequence. Thus, the term “operatively linked” or“operably associated” as used herein, refers to nucleotide sequences ona single nucleic acid molecule that are functionally associated. Thoseskilled in the art will appreciate that the control sequences (e.g.,promoter) need not be contiguous with the coding sequence, as long asthey functions to direct the expression thereof. Thus, for example,intervening untranslated, yet transcribed, sequences can be presentbetween a promoter and a coding sequence, and the promoter sequence canstill be considered “operably linked” to the coding sequence.

“Nucleotide sequence of interest” refers to any nucleotide sequencewhich, when introduced into a plant, confers upon the plant a desiredcharacteristic such as antibiotic resistance, virus resistance, insectresistance, disease resistance, or resistance to other pests, herbicidetolerance, abiotic stress resistance (e.g., drought tolerance, salttolerance, tolerance to waterlogging and/or submergence stress, and thelike), improved nutritional value, improved performance in an industrialprocess or altered reproductive capability. The “nucleotide sequence ofinterest” can encode a polypeptide or functional RNA (e.g., a regulatoryRNA). For example, the “nucleotide sequence of interest” may be one thatis transferred to plants for the production of a polypeptide (e.g., anenzyme, hormone, growth factor or antibody) for commercial production.

A “heterologous nucleotide sequence” or “heterologous nucleotidesequence of interest” as used herein is a coding sequence that isheterologous to the HaHB11 promoter of the invention (i.e., is not thenative HaHB11 sequence). The heterologous nucleotide sequence can encodea polypeptide or a functional RNA. A “heterologous promoter” is apromoter that is heterologous to the nucleotide sequence with which itis operatively associated. For example, according to the presentinvention, the HaHB11 coding sequence can be operatively associated witha heterologous promoter (e.g., a promoter that is not the native HaHB11promoter sequence with which the HaHB11 coding sequence is associated inits naturally occurring state).

By the term “express,” “expressing” or “expression” (or othergrammatical variants) of a nucleic acid coding sequence, it is meantthat the sequence is transcribed. In particular embodiments, the terms“express,” “expressing” or “expression” (or other grammatical variants)can refer to both transcription and translation to produce an encodedpolypeptide.

“Wild-type” nucleotide sequence or amino acid sequence refers to anaturally occurring (“native”) or endogenous nucleotide sequence(including a cDNA corresponding thereto) or amino acid sequence.

The terms “nucleic acid,” “polynucleotide” and “nucleotide sequence” canbe used interchangeably herein unless the context indicates otherwise.These terms encompass both RNA and DNA, including cDNA, genomic DNA,partially or completely synthetic (e.g., chemically synthesized) RNA andDNA, and chimeras of RNA and DI′JA. The nucleic acid, polynucleotide ornucleotide sequence may be double-stranded or single-stranded, andfurther may be synthesized using nucleotide analogs or derivatives(e.g., inosine or phosphorothioate nucleotides). Such nucleotides can beused, for example, to prepare nucleic acids, polynucleotides andnucleotide sequences that have altered base-pairing abilities orincreased resistance to nucleases. The present invention furtherprovides a nucleic acid, polynucleotide or nucleotide sequence that isthe complement (which can be either a full complement or a partialcomplement) of a nucleic acid, polynucleotide or nucleotide sequence ofthe invention (e.g., encodes a nucleic acid, polynucleotide ornucleotide sequence comprising, consisting essentially of, or consistingof a HaHB11 promoter element and/or is the complement of a HaHB11 codingsequence of the invention). Nucleotide sequences are presented herein bysingle strand only, in the 5′ to 3′ direction, from left to right,unless specifically indicated otherwise. Nucleotides and amino acids arerepresented herein in the manner recommended by the IUPAC-IUBBiochemical Nomenclature Commission, or (for amino acids) by either theone-letter code, or the three letter code, both in accordance with 37C.F.R. § 1.822 and established usage.

The nucleic acids and polynucleotides of the invention are optionallyisolated. An “isolated” nucleic acid molecule or polynucleotide is anucleic acid molecule or polynucleotide that, by the hand of man, existsapart from its native environment and is therefore not a product ofnature. An isolated nucleic acid molecule or isolated polynucleotide mayexist in a purified form or may exist in a non-native environment suchas, for example, a recombinant host cell. Thus, for example, the term“isolated” means that it is separated from the chromosome and/or cell inwhich it naturally occurs. A nucleic acid or polynucleotide is alsoisolated if it is separated from the chromosome and/or cell in which itnaturally occurs and is then inserted into a genetic context, achromosome, a chromosome location, and/or a cell in which it does notnaturally occur. The recombinant nucleic acid molecules andpolynucleotides of the invention can be considered to be “isolated.”

Further, an “isolated” nucleic acid or polynucleotide is a nucleotidesequence (e.g., DNA or RNA) that is not immediately contiguous withnucleotide sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The “isolated” nucleic acid orpolynucleotide can exist in a cell (e.g., a plant cell), optionallystably incorporated into the genome. According to this embodiment, the“isolated” nucleic acid or polynucleotide can be foreign to thecell/organism into which it is introduced, or it can be native to an thecell/organism (e.g., Helianthus annuus), but exist in a recombinant form(e.g., as a chimeric nucleic acid or polynucleotide) and/or can be anadditional copy of an endogenous nucleic acid or polynucleotide. Thus,an “isolated nucleic acid molecule” or “isolated polynucleotide” canalso include a nucleotide sequence derived from and inserted into thesame natural, original cell type, but which is present in a non-naturalstate, e.g., present in a different copy number, in a different geneticcontext and/or under the control of different regulatory sequences thanthat found in the native state of the nucleic acid molecule orpolynucleotide.

In representative embodiments, the “isolated” nucleic acid orpolynucleotide is substantially free of cellular material (includingnaturally associated proteins such as histones, transcription factors,and the like), viral material, and/or culture medium (when produced byrecombinant DNA techniques), or chemical precursors or other chemicals(when chemically synthesized). Optionally, in representativeembodiments, the isolated nucleic acid or polynucleotide is at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

As used herein, the term “recombinant” nucleic acid, polynucleotide ornucleotide sequence refers to a nucleic acid, polynucleotide ornucleotide sequence that has been constructed, altered, rearrangedand/or modified by genetic engineering techniques. The term“recombinant” does not refer to alterations that result from naturallyoccurring events, such as spontaneous mutations, or from non-spontaneousmutagenesis.

A “vector” is any nucleic acid molecule for the cloning of and/ortransfer of a nucleic acid into a cell. A vector may be a replicon towhich another nucleotide sequence may be attached to allow forreplication of the attached nucleotide sequence. A “replicon” can be anygenetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome)that functions as an autonomous unit of nucleic acid replication in thecell, i.e., capable of nucleic acid replication under its own control.The term “vector” includes both viral and nonviral (e.g., plasmid)nucleic acid molecules for introducing a nucleic acid into a cell invitro, ex vivo, and/or in vivo, and is optionally an expression vector.A large number of vectors known in the art may be used to manipulate,deliver and express polynucleotides. Vectors may be engineered tocontain sequences encoding selectable markers that provide for theselection of cells that contain the vector and/or have integrated someor all of the nucleic acid of the vector into the cellular genome. Suchmarkers allow identification and/or selection of host cells thatincorporate and express the proteins encoded by the marker. A“recombinant” vector refers to a viral or non-viral vector thatcomprises one or more heterologous nucleotide sequences (e.g.,transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Plant viralvectors that can be used include, but are not limited to, Agrobacteriumtumefaciens, Agrobacterium rhizogenes and geminivirus vectors. Non-viralvectors include, but are not limited to, plasmids, liposomes,electrically charged lipids (cytofectins), nucleic acid-proteincomplexes, and biopolymers. In addition to a nucleic acid of interest, avector may also comprise one or more regulatory regions, and/orselectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (e.g., delivery to specific tissues,duration of expression, etc.).

The term “fragment,” as applied to a nucleic acid or polynucleotide,will be understood to mean a nucleotide sequence of reduced lengthrelative to the reference or full-length nucleotide sequence andcomprising, consisting essentially of and/or consisting of contiguousnucleotides from the reference or full-length nucleotide sequence. Sucha fragment according to the invention may be, where appropriate,included in a larger polynucleotide of which it is a constituent. Insome embodiments, such fragments can comprise, consist essentially of,and/or consist of oligonucleotides having a length of at least about 8,10, 12, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 405, 410, 425,450, 455, 460, 475, 500, 505, 510, 515 or 520 nucleotides (optionally,contiguous nucleotides) or more from the reference or full-lengthnucleotide sequence, as long as the fragment is shorter than thereference or full-length nucleotide sequence. In representativeembodiments, the fragment is a biologically active nucleotide sequence,as that term is described herein.

A “biologically active” nucleotide sequence is one that substantiallyretains at least one biological activity normally associated with thewild-type nucleotide sequence, for example, the ability to drivetranscription of an operatively associated coding sequence. Inparticular embodiments, the “biologically active” nucleotide sequencesubstantially retains all of the biological activities possessed by theunmodified sequence. By “substantially retains” biological activity, itis meant that the nucleotide sequence retains at least about 50%, 60%,75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activityof the native nucleotide sequence (and can even have a higher level ofactivity than the native nucleotide sequence). For example, abiologically active promoter element is able to control, regulate and/orenhance the expression of a nucleotide sequence operably associated withthe promoter. Methods of measuring expression of a nucleotide sequenceare well known in the art and include Northern blots, RNA run-on assaysand methods of measuring the presence of an encoded polypeptide (e.g.,antibody based methods or visual inspection in the case of a reporterpolypeptide).

Two nucleotide sequences are said to be “substantially identical” toeach other when they share at least about 60%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, 99% or even 100% sequence identity.

Two amino acid sequences are said to be “substantially identical” or“substantially similar” to each other when they share at least about60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or even 100% sequenceidentity or similarity, respectively.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or polypeptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids.

As used herein “sequence similarity” is similar to sequence identity (asdescribed herein), but permits the substitution of conserved amino acids(e.g., amino acids whose side chains have similar structural and/orbiochemical properties), which are well-known in the art.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid has sequence identity or an amino acidsequence has sequence identity or similarity to a known sequence.Sequence identity or similarity may be determined using standardtechniques known in the art, including, but not limited to, the localsequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482(1981), by the sequence identity alignment algorithm of Needleman etal., J. Mol. Biol. 48,443 (1970), by the search for similarity method ofPearson & Lipman, PNAS 85, 2444 (1988), by computerized implementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12:387-395 (1984), preferably using the defaultsettings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng et al., J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins etal., CABIOS 5:151-153 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215:403-410, (1990) and Karlin et al.,PNAS 90:5873-5787 (1993). A particularly useful BLAST program is theWU-BLAST-2 program which was obtained from Altschul et al., Methods inEnzymology, 266, 460-480 (1996); blast.wustl/edu/blastl READMEhtml.WU-BLAST-2 uses several search parameters, which are preferably set tothe default values. The parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucleic Acids Res. 25:3389-3402 (1997).

The CLUSTAL program can also be used to determine sequence similarity.This algorithm is described by Higgins et al., Gene 73:237 (1988);Higgins et al., CABIOS 5:151-153 (1989); Corpet et al., Nucleic AcidsRes. 16:10881-90 (1988); Huang et al., CABIOS 8:155-65 (1992); andPearson et al., Meth. Mol. Biol. 24:307-331 (1994).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer nucleotides than the nucleic acids disclosed herein, it isunderstood that in one embodiment, the percentage of sequence identitywill be determined based on the number of identical nucleotides acids inrelation to the total number of nucleotide bases. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of nucleotidebases in the shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

Two nucleotide sequences can also be considered to be substantiallyidentical when the two sequences hybridize to each other under stringentconditions. A nonlimiting example of “stringent” hybridizationconditions include conditions represented by a wash stringency of 50%Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C.“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. An extensiveguide to the hybridization of nucleic acids is found in Tij ssenLaboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York (1993). In some representativeembodiments, two nucleotide sequences considered to be substantiallyidentical hybridize to each other under highly stringent conditions.Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH.

As used herein, the term “polypeptide” encompasses both peptides andproteins (including fusion proteins), unless indicated otherwise.

A “fusion protein” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame.

The polypeptides of the invention are optionally “isolated.” An“isolated” polypeptide is a polypeptide that, by the hand of man, existsapart from its native environment and is therefore not a product ofnature. An isolated polypeptide may exist in a purified form or mayexist in a non-native environment such as, for example, a recombinanthost cell. The recombinant polypeptides of the invention can beconsidered to be “isolated.”

In representative embodiments, an “isolated” polypeptide means apolypeptide that is separated or substantially free from at least someof the other components of the naturally occurring organism or virus,for example, the cell or viral structural components or otherpolypeptides or nucleic acids commonly found associated with thepolypeptide. In particular embodiments, the “isolated” polypeptide is atleast about 1%, 5%, 10%, 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99% or more pure (w/w). In other embodiments, an “isolated”polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold,100-fold, 1000-fold, 10,000-fold, or more enrichment of the protein(w/w) is achieved as compared with the starting material. Inrepresentative embodiments, the isolated polypeptide is a recombinantpolypeptide produced using recombinant nucleic acid techniques. Inembodiments of the invention, the polypeptide is a fusion protein.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid of reduced length relative to a reference polypeptideor the full-length polypeptide (e.g., HaHB11) and comprising, consistingessentially of, and/or consisting of a sequence of contiguous aminoacids from the reference or full-length polypeptide. Such a fragmentaccording to the invention may be, where appropriate, included as partof a fusion protein of which it is a constituent. In some embodiments,such fragments can comprise, consist essentially of, and/or consist ofpolypeptides having a length of at least about 50, 75, 100, 125, 150,160, 165, 170, 171, 172, 173 or 174 amino acids (optionally, contiguousamino acids) from the reference or full-length polypeptide, as long asthe fragment is shorter than the reference or full-length polypeptide.In representative embodiments, the fragment is biologically active, asthat term is defined herein.

A “biologically active” polypeptide is one that substantially retains atleast one biological activity normally associated with the wild-typepolypeptide, for example, the ability to increase tolerance to abioticstress and/or increase yield. In another embodiment, a biologicallyactive polypeptide is capable of: binding the sequence CAAT(A/T)ATTG(SEQ ID NO:7) under physiological conditions in vitro, binding one ormore different endogenous host plant proteins under physiologicalconditions in vitro, and/or activating transcription in a yeasttwo-hybrid system such as, described herein. In particular embodiments,the “biologically active” polypeptide substantially retains all of thebiological activities possessed by the unmodified (wild-type) sequence.By “substantially retains” biological activity, it is meant that thepolypeptide retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%,98%, 99%, or more, of the biological activity of the native polypeptide(and can even have a higher level of activity than the nativepolypeptide). Methods of measuring yield and tolerance to abiotic stressare known in the art, with non-limiting and exemplary methods aredescribed in the working Examples herein.

“Introducing” in the context of a plant cell, plant tissue, plant partand/or plant means contacting a nucleic acid molecule with the plantcell, plant tissue, plant part, and/or plant in such a manner that thenucleic acid molecule gains access to the interior of the plant cell ora cell of the plant tissue, plant part or plant. Where more than onenucleic acid molecule is to be introduced, these nucleic acid moleculescan be assembled as part of a single polynucleotide or nucleic acidconstruct, or as separate polynucleotide or nucleic acid constructs, andcan be located on the same or different nucleic acid constructs.Accordingly, these nucleic acid molecules can be introduced into plantcells in a single transformation event, in separate transformationevents, or, e.g., as part of a breeding protocol.

The term “transformation” as used herein refers to the introduction of aheterologous nucleic acid into a cell. Transformation of a cell may bestable or transient. Thus, a transgenic plant cell, plant tissue, plantpart and/or plant of the invention can be stably transformed ortransiently transformed.

“Transient transformation” in the context of a polynucleotide means thata polynucleotide is introduced into the cell and does not integrate intothe genome of the cell.

As used herein, “stably introducing,” “stably introduced,” “stabletransformation” or “stably transformed” (and similar terms) in thecontext of a polynucleotide introduced into a cell, means that theintroduced polynucleotide is stably integrated into the genome of thecell (e.g., into a chromosome or as a stable-extra-chromosomal element).As such, the integrated polynucleotide is capable of being inherited byprogeny cells and plants.

“Genome” as used herein includes the nuclear and/or plastid genome, andtherefore includes integration of a polynucleotide into, for example,the chloroplast genome. Stable transformation as used herein can alsorefer to a polynucleotide that is maintained extrachromosomally, forexample, as a minichromosome.

As used herein, the terms “transformed” and “transgenic” refer to anyplant, plant cell, plant tissue (including callus), or plant part thatcontains all or part of at least one recombinant or isolated nucleicacid, polynucleotide or nucleotide sequence. In representativeembodiments, the recombinant or isolated nucleic acid, polynucleotide ornucleotide sequence is stably integrated into the genome of the plant(e.g., into a chromosome or as a stable extra-chromosomal element), sothat it is passed on to subsequent generations of the cell or plant.

The term “plant part,” as used herein, includes but is not limited toreproductive tissues (e.g., petals, sepals, stamens, pistils,receptacles, anthers, pollen, flowers, fruits, flower bud, ovules,seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues(e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles,stalks, shoots, branches, bark, apical meristem, axillary bud,cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem andxylem); specialized cells such as epidermal cells, parenchyma cells,chollenchyma cells, schlerenchyma cells, stomates, guard cells, cuticle,mesophyll cells; callus tissue; and cuttings. The term “Plant part” alsoincludes plant cells including plant cells that are intact in plantsand/or parts of plants, plant protoplasts, plant tissues, plant organsplant cell tissue cultures, plant calli, plant clumps, and the like. Asused herein, “shoot” refers to the above ground parts including theleaves and stems.

The term “tissue culture” encompasses cultures of tissue, cells,protoplasts and callus.

As used herein, “plant cell” refers to a structural and physiologicalunit of the plant, which typically comprise a cell wall but alsoincludes protoplasts. A plant cell of the present invention can be inthe form of an isolated single cell or can be a cultured cell or can bea part of a higher-organized unit such as, for example, a plant tissue(including callus) or a plant organ. Any plant (or groupings of plants,for example, into a genus or higher order classification) can beemployed in practicing the present invention including angiosperms orgymnosperms, monocots or dicots.

Exemplary transgenic plants of the invention include, but are notlimited to, corn (Zea mays), canola (Brassica napus, Brassica rapassp.), alfalfa (Medicago saliva), rice (Oryza sativa), rape (Brassicanapus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tobacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), apple (Malus pumila), blackberry (Rubus),strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera),apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica),plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullusvulgaris), duckweed (Lemna), oats, barley, vegetables, ornamentals,conifers, and turfgrasses (e.g., for ornamental, recreational or foragepurposes), and biomass grasses (e.g., switchgrass and miscanthus).

Exemplary transgenic vegetables of the invention include, but are notlimited to, Solanaceous species (e.g., tomatoes; Lycopersiconesculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota),cauliflower (Brassica oleracea), celery (apium graveolens), eggplant(Solanum melongena), asparagus (Asparagus officinalis), ochra(Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), members of the genusCucurbita such as Hubbard squash (C. Hubbard), Butternut squash (C.moschata), Zucchini (C. pepo), Crookneck squash (C. crookneck), C.argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis,C. foetidissima, C. lundelliana, and C. martinezii, and members of thegenus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C.cantalupensis), and musk melon (C. melo). Ornamentals include azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(dianthus caryophyllus), poinsettia (Euphorbia pulcherima), andchrysanthemum.

Conifers, which may be employed in practicing the present invention,include, for example pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis).

Turfgrass which may be employed in practicing the present invention,include but are not limited to zoysiagrasses, bentgrasses, fescuegrasses, bluegrasses, St. Augustinegrasses, bermudagrasses,bufallograsses, ryegrasses, and orchardgrasses.

In particular embodiments, the transgenic plants of the invention are amember selected from wheat (Tritium aestivum), corn (Zea mays) and rice(Oryza sativa). In an additional embodiment, the transgenic plants arealfalfa or sunflower. In a particular embodiment, the transgenic plantsare soybean (Glycine max).

Also included as transgenic plants of the invention are plants thatserve primarily as laboratory models, e.g., Arabidopsis.

II. HaHB11 Polypeptides, HaHB11 Coding Sequences and Promoter Sequences

As one aspect, the present invention provides HaHB11 polypeptides. Theterm “HaHB11 polypeptide” is intended to encompass the HaHB11polypeptides specifically described herein (e.g., SEQ ID NO:3 and SEQ IDNO:10) as well as equivalents thereof, e.g., that have substantiallyidentical or similar amino acid sequences (as described herein) to theHaHB11 polypeptides specifically described herein, optionallybiologically active equivalents that have one or more of the biologicalactivities of the HaHB11 polypeptides specifically described herein. Theterm “HaHB11 polypeptide” also encompasses fragments of the full-lengthHaHB11 polypeptides specifically disclosed herein, optionallybiologically active fragments, and biologically active equivalentsthereof that have substantially identical or similar amino acidsequences to a fragment of a full-length HaHB11 polypeptide specificallydisclosed herein. Further, the term “HaHB11 polypeptide” includessequences from Helianthus annuus or can be an ortholog from any othersuitable plant species and also includes naturally occurring allelicvariations, isoforms, splice variants and the like. Exemplary HaHB11polypeptides of the invention include, but are not limited to, a proteinhaving the amino acid sequence of: SEQ ID NO:11, SEQ ID NO:12; SEQ IDNO:13, SEQ ID NO:14 or SEQ ID NO:15. The HaHB11 polypeptide sequencescan further be wholly or partially synthetic.

Biological activities associated with expression of HaHB11 in a plantinclude but are not limited to, increased tolerance to abiotic stress(e.g., drought, submergence, desubmergence, waterlogging and/or saltstress), delayed development, greater or reduced plant height, and/orincreased yield (e.g., under normal conditions and/or conditions of mildand/or severe abiotic stress). Additional biological activitiesassociated with expression of HaHB11 in a plant include increased rootlength. Methods of assessing tolerance to abiotic stress, development,and yield are well known in the art (see the Examples for exemplarymethods).

In particular embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of an isolated HaHB11 polypeptide of SEQ IDNO:3 or an equivalent thereof (including fragments and equivalentsthereof).

In additional particular embodiments, the HaHB11 polypeptide comprises,consists essentially of, or consists of an isolated HaHB11 polypeptideof SEQ ID NO:10 or an equivalent thereof (including fragments andequivalents thereof).

In some embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of an the N terminal region, homeodomain,leucine zipper or C-terminal region of a HaHB11 polypeptide disclosedherein.

In particular embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of an N terminal region of a HaHB11polypeptide disclosed herein. In some embodiments, the HaHB11polypeptide comprises, consists essentially of, or consists of an Nterminal region having the amino acid sequence of amino acids 1 to 14 ofSEQ ID NO:3. In some embodiments, the HaHB11 polypeptide comprises,consists essentially of, or consists of an N terminal region having anamino acid sequence selected from: amino acids 1 to 14 of SEQ ID NO:12,amino acids 1 to 14 of SEQ ID NO:14; and amino acids 1 to 14 of SEQ IDNO:15.

In additional embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of a homeodomain of a HaHB11 polypeptidedisclosed herein. In some embodiments, the HaHB11 polypeptide comprises,consists essentially of, or consists of a homeodomain having the aminoacid sequence of amino acids 15 to 74 of SEQ ID NO:3. In someembodiments, the HaHB11 polypeptide comprises, consists essentially of,or consists of a homeodomain having the amino acid sequence of aminoacids 15 to 77 of SEQ ID NO:10. In some embodiments, the HaHB11polypeptide comprises, consists essentially of, or consists of ahomeodomain having an amino acid sequence of amino acids 15 to 74 of SEQID NO:12 or amino acids 15 to 74 of SEQ ID NO:13.

In additional embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of a leucine zipper of a HaHB11 polypeptidedisclosed herein. In some embodiments, the HaHB11 polypeptide comprises,consists essentially of, or consists of a leucine zipper having theamino acid sequence of amino acids 75-110 of SEQ ID NO:3. In someembodiments, the HaHB11 polypeptide comprises, consists essentially of,or consists of a leucine zipper having the amino acid sequence of aminoacids 78-113 of SEQ ID NO:11. In some embodiments, the HaHB11polypeptide comprises, consists essentially of, or consists of a leucinezipper having an amino acid sequence selected from: amino acids 77 to112 of SEQ ID NO:14 or amino acids 76 to 111 of SEQ ID NO:15.

In additional embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of a C-terminal region of a HaHB11polypeptide disclosed herein. In some embodiments, the HaHB11polypeptide comprises, consists essentially of, or consists of a leucinezipper having the amino acid sequence of amino acids 111-175 of SEQ IDNO:3. In some embodiments, the HaHB11 polypeptide comprises, consistsessentially of, or consists of a leucine zipper having the amino acidsequence of amino acids 114-181 of SEQ ID NO:10. In some embodiments,the HaHB11 polypeptide comprises, consists essentially of, or consistsof a C-terminal region having an amino acid sequence selected from:amino acids 114 to 180 of SEQ ID NO:11, amino acids 111 to 179 of SEQ IDNO:12; amino acids 111 to 177 of SEQ ID NO:13, amino acids 113 to 183 ofSEQ ID NO:14 and amino acids 112 to 186 of SEQ ID NO:15.

Equivalents of the HaHB11 polypeptides of the invention encompass thosethat have substantial amino acid sequence identity or similarity, forexample, at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or more amino acid sequence identity or similarity with the amino acidsequences specifically disclosed herein (e.g., SEQ ID NO:3) or afragment thereof, optionally a biologically active fragment. Additionalequivalents of the HaHB11 polypeptides of the invention encompass thosethat have substantial amino acid sequence identity or similarity, forexample, at least about 96%, 97%, 98%, 99% or more amino acid sequenceidentity or similarity with the amino acid sequence of SEQ ID NO:10 or afragment thereof, optionally a biologically active fragment.

In representative embodiments, the HaHB11 polypeptide comprises thehomeodomain, leucine zipper, the transactivation site, and/or theputative protein-kinase phosphorylation site domains (see, schematic inFIG. 1B), and optionally any sequence variability occurs outside of thisregion(s). In representative embodiments, the HaHB11 polypeptidecomprises the first, second, third and/or fourth casein-phosphorylationsite (numbering with respect to the N-terminus; FIG. 1B), and optionallyany variability occurs outside this domain. In representativeembodiments, the HaHB11 polypeptide comprises the N-terminal region (seeFIG. 1B) and, optionally, any sequence variation occurs outside thisdomain. In additional representative embodiments, the HaHB11 polypeptidecomprises the C-terminal domain (FIG. 1B) and; optionally, any sequencevariability occurs outside of this region. In representativeembodiments, the homeodomain, leucine zipper, transactivation site,protein kinase phosphorylation site, casein phosphorylation sites,N-terminal domain and/or C-terminal domain have the sequence(s) as shownin FIG. 1B. In additional embodiments, the sequence is an equivalent ofthe sequence as shown in FIG. 1B (e.g., substantially similar oridentical). In representative embodiments, the HaHB11 polypeptide doesnot comprise the N-terminal domain, the homeodomain and/or the leucinezipper domain (the location of these domains is shown in FIG. 1B). Inembodiments, the HaHB11 polypeptide binds in vitro and/or in vivo to thepseudopalindromic sequence CAAT(A/T)ATTG (SEQ ID NO:7).

In representative embodiments, the HaHB11 polypeptide comprises thehomeodomain, leucine zipper, the transactivation site, and/or putativeprotein-kinase phosphorylation site domains (see, schematic in FIG. 1B),

Unless indicated otherwise, the HaHB11 polypeptide can be a fusionprotein. For example, it may be useful to express the HaHB11polypeptides as a fusion protein that can be recognized by acommercially available antibody (e.g., FLAG motifs) or as a fusionprotein that can otherwise be more easily purified (e.g., by addition ofa poly-His tail). Additionally, fusion proteins that enhance thestability of the protein may be produced, e.g., fusion proteinscomprising maltose binding protein (MBP) or glutathione-S-transferase.As another alternative, the fusion protein can comprise a reportermolecule. HaHB11 is a transcription factor and HaHB11 fusion proteinscan also be generated for use in yeast two-hybrid systems (e.g.,GAL4-HaHB11 fusions), as is known in the art.

It will further be understood that the HaHB11 polypeptides specificallydisclosed herein will typically tolerate substitutions in the amino acidsequence and substantially retain biological activity. To routinelyidentify biologically active HaHB11 polypeptides of the invention otherthan those specifically disclosed herein, amino acid substitutions maybe based on any characteristic known in the art, including the relativesimilarity or differences of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. In particular embodiments, conservative substitutions (i.e.,substitution with an amino acid residue having similar properties) aremade in the amino acid sequence encoding the HaHB11 polypeptide.

In making amino acid substitutions, the hydropathic index of amino acidscan be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (see, Kyte et al., J. Mol. Biol. 157:105 (1982)).It is accepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte et al., Id.), andthese are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5). It is also understood in the artthat the substitution of amino acids can be made on the basis ofhydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest localaverage hydrophilicity of a protein, as governed by the hydrophilicityof its adjacent amino acids, correlates with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

The HaHB11 polypeptides of the present invention also encompass HaHB11polypeptide fragments (optionally, biologically active HaHB11fragments), and equivalents thereof (optionally, biologically activeequivalents). The length of the HaHB11 fragment is not critical.Illustrative functional HaHB11 protein fragments comprise at least about50, 75, 100, 125, 150, 160, 165, 170, 171, 172, 173 or 174 amino acids(optionally, contiguous amino acids) of a HaHB11 polypeptide. Inrepresentative embodiments, the HaHB11 polypeptide comprises thehomeodomain, leucine zipper, the transactivation site, and/or theputative protein-kinase phosphorylation site domains (see, schematic inFIG. 1B), and optionally any sequence variability occurs outside of thisregion(s). In representative embodiments, the HaHB11 comprises thefirst, second, third and/or fourth casein-phosphorylation site(numbering with respect to the N-terminus; FIG. 1B), and optionally anyvariability occurs outside this domain. In representative embodiments,the HaHB11 polypeptide comprises the N-terminal region (see FIG. 1B)and, optionally, any sequence variation occurs outside this domain. Inadditional representative embodiments, the HaHB11 polypeptide comprisesthe C-terminal domain (FIG. 1B) and; optionally, any sequencevariability occurs outside of this region. In representativeembodiments, the homeodomain, leucine zipper, transactivation site,protein kinase phosphorylation site, casein phosphorylation sites,N-terminal domain and/or C-terminal domain have the sequence(s) as shownin FIG. 1B. In additional embodiments, the sequence is an equivalent ofthe sequence as shown in FIG. 1B (e.g., substantially similar oridentical). In representative embodiments, the HaHB11 polypeptide doesnot comprise the N-terminal domain, the homeodomain and/or the leucinezipper domain (location of these domains shown in FIG. 1B). Inembodiments, the HaHB11 polypeptide binds in vitro and/or in vivo to thepseudopalindromic sequence CAAT(A/T)ATTG (SEQ ID NO:7).

In representative embodiments, equivalents of the HaHB11 polypeptides(including variants and fragments) retain one, two, three, four or allfive of the leucines in the leucine zipper region of SEQ ID NO:3 (see,FIG. 1B).

In representative embodiments, equivalents of the HaHB11 polypeptides(including variants and fragments) retain one, two, three, four or allfive of the leucines in the leucine zipper region of SEQ ID NO:10 (aminoacid residues 111-175 of SEQ ID NO:10).

In representative embodiments, the invention provides an isolated HaHB11polypeptide comprising, consisting essentially of, or consisting of anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of SEQ ID NO:3; (b) an amino acid sequence having at leastabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more aminoacid sequence identity or similarity with the amino acid sequence of SEQID NO:3, optionally wherein the HaHB11 polypeptide is biologicallyactive; and (c) a fragment of at least about 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 155, 160, 171, 172, 173 or 174 amino acids ofthe amino acid sequence of (a) or (b) above, optionally wherein thefragment is biologically active.

In representative embodiments, the invention provides an isolated HaHB11polypeptide comprising, consisting essentially of, or consisting of anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of SEQ ID NO:10; (b) an amino acid sequence having atleast about 96%, 97%, 98%, 99% or more amino acid sequence identity orsimilarity with the amino acid sequence of SEQ ID NO:10, optionallywherein the HaHB11 polypeptide is biologically active; and (c) afragment comprising amino acids 15 to 30, or 145 to 160 of SEQ ID NO:10that is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,155, 160, 171, 172, 173 or 174 amino acids of the amino acid sequence of(a) or (b) above, optionally wherein the fragment is biologicallyactive.

In representative embodiments, the invention provides an isolated HaHB11polypeptide comprising, consisting essentially of, or consisting of anamino acid sequence selected from the group consisting of: (a) the aminoacid sequence of SEQ ID NO:11, SEQ ID NO:12; SEQ ID NO:13, SEQ ID NO:14,or SEQ ID NO:15; (b) an amino acid sequence having at least about 96%,97%, 98%, 99% or more amino acid sequence identity or similarity withthe amino acid sequence of SEQ ID NO:11, SEQ ID NO:12; SEQ ID NO:13, SEQID NO:14, or SEQ ID NO:15, optionally wherein the HaHB11 polypeptide isbiologically active; and (c) a fragment comprising amino acids 140 to155 of SEQ ID NO:11, amino acids 140 to 155 of SEQ ID NO:12, amino acids150 to 165 of SEQ ID NO:13, amino acids 80 to 95 of SEQ ID NO:14, aminoacids 150 to 165 of SEQ ID NO:14, amino acids 125 to 140 of SEQ IDNO:15, or amino acids 150 to 165 of SEQ ID NO:15, that is at least aboutat least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 155,160, 171, 172, 173 or 174 amino acids of the amino acid sequence of (a)or (b) above, optionally wherein the fragment is biologically active.

The invention further provides antibodies and antibody fragments thatspecifically bind to the HaHB11 polypeptides of the invention.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including for example mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric, humanized or human antibody. See, e.g., Walker etal., Molec. Immunol. 26:403-411 (1989). The antibodies can berecombinant monoclonal antibodies produced according to the methodsdisclosed in U.S. Pat. No. 4,474,893 or 4,816,567. The antibodies canalso be chemically constructed according to the method disclosed in U.S.Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., Science254:1275-1281 (1989)).

Monoclonal antibodies according to the present invention can be producedin a hybridoma cell line according to the technique of Kohler et al.,Nature 265:495-97 (1975). For example, a solution containing theappropriate antigen can be injected into a mouse and, after a sufficienttime, the mouse sacrificed and spleen cells obtained. The spleen cellsare then immortalized by fusing them with myeloma cells or with lymphomacells, typically in the presence of polyethylene glycol, to producehybridoma cells. The hybridoma cells are then grown in a suitable mediumand the supernatant screened for monoclonal antibodies having thedesired specificity. Monoclonal Fab fragments can be produced in E. coliby recombinant techniques known to those skilled in the art. See, e.g.,Huse, Science 246:1275-1281 (1989).

Antibodies specific to a target polypeptide can also be obtained byphage display techniques known in the art.

The invention also provides nucleic acids encoding the HaHB11polypeptides of the invention, optionally a biologically active HaHB11polypeptide. The nucleic acid can be from any plant species of origin(e.g., Helianthus annuus) or can be partially or completely synthetic.In representative embodiments, the nucleic acid encoding the HaHB11polypeptide is an isolated nucleic acid.

HaHB11 orthologs from other organisms, in particular other plants, canbe routinely identified using methods known in the art (e.g., orthologsfrom species belonging to the Asteraceae family [also known as theCompositae family], such as a species of lettuce). For example, PCR andother amplification techniques and hybridization techniques can be usedto identify such orthologs based on their sequence similarity to thesequences set forth herein.

In representative embodiments, the invention encompasses polynucleotidesencoding the HaHB11 polypeptides of the invention having substantialnucleotide sequence identity with the polynucleotides specificallydisclosed herein encoding HaHB11 (e.g., SEQ ID NO:1, nucleotides 7-944of SEQ ID NO:1, and SEQ ID NO:2), or fragments thereof, and which encodea HaHB11 polypeptide (including fragments), optionally a biologicallyactive HaHB11 polypeptide. In some embodiments, the polynucleotidesencoding the HaHB11 polypeptides have substantial nucleotide sequenceidentity with the polynucleotides of SEQ ID NO:9, or fragments thereof,and which encode a HaHB11 polypeptide (including fragments), optionallya biologically active HaHB11 polypeptide.

The invention also provides polynucleotides encoding the HaHB11polypeptides of the invention, wherein the polynucleotide hybridizes tothe complete complement of the HaHB11 nucleic acid sequencesspecifically disclosed herein (e.g., SEQ ID NO:1, nucleotides 7-944 ofSEQ ID NO:1, and SEQ ID NO:2), or fragments thereof, under stringenthybridization conditions as known by those skilled in the art and encodea HaHB11 polypeptide (including fragments), optionally a biologicallyactive HaHB11 polypeptide. In some embodiments, the polynucleotidehybridizes to nucleotides 50 to 70, 430 to 450, 600 to 620, or 731 to751 of SEQ ID NO:9, or fragments thereof, under stringent hybridizationconditions and encode a HaHB11 polypeptide (including fragments),optionally a biologically active HaHB11 polypeptide. Further, it will beappreciated by those skilled in the art that there can be variability inthe polynucleotides that encode the HaHB11 polypeptides of the presentinvention due to the degeneracy of the genetic code. The degeneracy ofthe genetic code, which allows different nucleotide sequences to codefor the same protein, is well known in the art. Moreover, plant orspecies-preferred codons can be used in the polynucleotides encoding theHaHB11 polypeptides of the invention, as is also well-known in the art.

The invention also provides polynucleotides encoding fragments of afull-length HaHB11 polypeptide, optionally biologically activefragments. Exemplary polynucleotides encoding HaHB11 fragments compriseat least about at least about 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 505, 510, 515 or 520or more nucleotide bases (optionally, contiguous bases) of apolynucleotide encoding a full-length HaHB11 polypeptide.

In exemplary, but non-limiting, embodiments, the invention provides anucleic acid (e.g., recombinant or isolated) comprising, consistingessentially of, or consisting of a nucleotide sequence encoding a HaHB11polypeptide, the nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence comprising the nucleotide sequence of SEQID NO:1, nucleotides 7-944 of SEQ ID NO:1, or SEQ ID NO:2; (b) anucleotide sequence comprising at least about 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,505, 510, 515 or 520 or more consecutive nucleotides of the nucleotidesequence of SEQ ID NO:1, nucleotides 7-944 of SEQ ID NO:1, or SEQ IDNO:2 (e.g., encoding a fragment, optionally a functional fragment of SEQID NO:3); (c) a nucleotide sequence having at least about 60%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to thenucleotide sequence of (a) or (b); (d) a nucleotide sequence thathybridizes to the complete complement of the nucleotide sequence of (a)or (b) under stringent hybridization conditions; and (e) a nucleotidesequence that differs from the nucleotide sequence of any of (a) to (d)due to the degeneracy of the genetic code. In representativeembodiments, the nucleotide sequence encodes a biologically activeHaHB11 polypeptide (including biologically active fragments of afull-length HaHB11 polypeptide).

In additional exemplary, but non-limiting, embodiments, the inventionprovides a nucleic acid (e.g., recombinant or isolated) comprising,consisting essentially of, or consisting of a nucleotide sequenceencoding a HaHB11 polypeptide, the nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence comprising the nucleotidesequence of SEQ ID NO:9 or nucleotides 174 to 190, 550 to 575, 725 to740, or 830 to 860 of SEQ ID NO:9; (b) a nucleotide sequence comprisingat least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 505, 510, 515 or 520 or moreconsecutive nucleotides of the nucleotide sequence of SEQ ID NO:9 thatinclude at least one sequence selected from nucleotides 50 to 70, 430 to450, 600 to 620 or 731 to 751 of SEQ ID NO:9 (e.g., encoding a fragment,optionally a functional fragment of SEQ ID NO:9); (c) a nucleotidesequence having at least about 90%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the nucleotide sequence of (a) or (b); and (d) anucleotide sequence that differs from the nucleotide sequence of any of(a) to (c) due to the degeneracy of the genetic code. In representativeembodiments, the nucleotide sequence encodes a biologically activeHaHB11 polypeptide (including biologically active fragments of afull-length HaHB11 polypeptide).

In representative embodiments, the nucleotide sequence encodes thepolypeptide of SEQ ID NO:3, or an equivalent polypeptide havingsubstantial amino acid sequence identity or similarity with SEQ ID NO:3(optionally, a biologically active equivalent). In representativeembodiments, the nucleotide sequence encodes an equivalent (optionally,a biologically active equivalent) of the polypeptide of SEQ ID NO:3 andhybridizes to the complete complement of nucleotide sequence of SEQ IDNO:1, nucleotides 7-944 of SEQ ID NO:1, or SEQ ID NO:2 under stringenthybridization conditions.

In additional representative embodiments, the nucleotide sequenceencodes the polypeptide of SEQ ID NO:10, or an equivalent polypeptidehaving substantial amino acid sequence identity or similarity with SEQID NO:10 (optionally, a biologically active equivalent). Inrepresentative embodiments, the nucleotide sequence encodes anequivalent (optionally, a biologically active equivalent) of thepolypeptide of SEQ ID NO:10 and hybridizes to the complete complement ofnucleotide sequence of nucleotides 50 to 70, 430 to 450, 600 to 620, or731 to 751 of SEQ ID NO:9, under stringent hybridization conditions.

In representative embodiments, the nucleotide sequence encodes thepolypeptide of SEQ ID NO:3. According to this embodiment, the nucleotidesequence can comprise, consist essentially of, or consist of SEQ IDNO:1, nucleotides 7-944 of SEQ ID NO:1, or SEQ ID NO:2. Also accordingto this embodiment, the nucleotide sequence can comprise, consistessentially of, or consist of a sequence that is distinct from that ofSEQ ID NO:2, but encodes the polypeptide of SEQ ID NO:3 due to thedegeneracy of the genetic code. In representative embodiments, thenucleotide sequence encodes a biologically active HaHB11 polypeptide Inother representative embodiments, the nucleotide sequence encodes thepolypeptide of SEQ ID NO:10. Also according to this embodiment, thenucleotide sequence can comprise, consist essentially of, or consist ofa sequence that is distinct from that of SEQ ID NO:9, but encodes thepolypeptide of SEQ ID NO:10 due to the degeneracy of the genetic code.In other representative embodiments, the nucleotide sequence encodes thepolypeptide of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 orSEQ ID NO:15.

Those skilled in the art will appreciate that both the homeodomain andleucine zipper motifs (HD-Zip) are functionally conserved andinterchangeable among HD-Zip I proteins. Accordingly, in representativeembodiments, the HaHB11 polypeptide comprises (i) a HD-Zip domain (e.g.,from an HD-Zip protein) that optionally binds CAAT(A/T)ATTG (SEQ IDNO:7) and (ii) amino acids 111 to 175 of SEQ ID NO:3 or an amino acidsequence that is substantially similar or identical thereto. Inembodiments of the invention, one, two three, four or all five of theleucines in the leucine zipper region of SEQ ID NO:3 (see, FIG. 1B) areconserved. Optionally, the HaHB11 polypeptide further comprises aminoacids 1 to 14 of SEQ ID NO:3 or an amino acid sequence that issubstantially similar or identical thereto. In embodiments of theinvention, the HaHB11 polypeptide is biologically active.

In additional representative embodiments, the HaHB11 polypeptidecomprises (i) a HD-Zip domain (e.g., from an HD-Zip protein) thatoptionally binds CAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 114 to181 of SEQ ID NO:10 or an amino acid sequence that is substantiallysimilar or identical thereto. In embodiments of the invention, one, twothree, four or all five of the leucines in the leucine zipper region ofSEQ ID NO:10 are conserved. Optionally, the HaHB11 polypeptide furthercomprises amino acids 1 to 14 of SEQ ID NO:10 or an amino acid sequencethat is substantially similar or identical thereto. In embodiments ofthe invention, the HaHB11 polypeptide is biologically active.

In additional representative embodiments, the HaHB11 polypeptidecomprises (i) a HD-Zip domain (e.g., from an HD-Zip protein) thatoptionally binds CAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids aminoacids 114 to 180 of SEQ ID NO:11, amino acids 111 to 179 of SEQ IDNO:12; amino acids 111 to 177 of SEQ ID NO:13, amino acids 113 to 183 ofSEQ ID NO:14, amino acids 112 to 186 of SEQ ID NO:15, or an amino acidsequence that is substantially similar or identical thereto. Inembodiments of the invention, one, two three, four or all five of theleucines in the leucine zipper region of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:15, respectively, are conserved.Optionally, the HaHB11 polypeptide further comprises amino acids 1 to 14of SEQ ID NO:12, amino acids 1 to 14 of SEQ ID NO:14 or amino acids 1 to14 of SEQ ID NO:15 or an amino acid sequence that is substantiallysimilar or identical thereto. In embodiments of the invention, theHaHB11 polypeptide is biologically active.

In further representative embodiments, the HaHB11 polypeptide comprises(i) a HD-Zip domain having an amino acid sequence selected from: aminoacids amino acids 15 to 110 of SEQ ID NO:3, 15 to 113 of SEQ ID NO:10,15 to 113 of SEQ ID NO:11, amino acids 15 to 110 of SEQ ID NO:12; aminoacids 15 to 110 of SEQ ID NO:13, amino acids 15 to 112 of SEQ ID NO:14and amino acids 15 to 111 of SEQ ID NO:15, or an amino acid sequencethat is substantially similar or identical thereto.

In other representative embodiments, the homeodomain of the HaHB11protein (e.g., amino acids 15 to 74 or SEQ ID NO:3 or an equivalentthereof) is replaced with a heterologous homeodomain. For example, theHaHB11 polypeptide can comprise (i) a HD domain (e.g., from an HD-Zipprotein); and (ii) amino acids 75 to 175 of SEQ ID NO:3 or an amino acidsequence that is substantially similar or identical to amino acids 75 to175 of SEQ ID NO:3, wherein the HaHB11 polypeptide optionally bindsCAAT(A/T)ATTG (SEQ ID NO:7). In another example, the HaHB11 polypeptidecan comprise (i) a HD domain (e.g., from an HD-Zip protein); and (ii)amino acids 78 to 181 of SEQ ID NO:10 or an amino acid sequence that issubstantially similar or identical to amino acids 78 to 181 of SEQ IDNO:10, wherein the HaHB11 polypeptide optionally binds CAAT(A/T)ATTG(SEQ ID NO:7). In another example, the HaHB11 polypeptide can comprise(i) a HD domain (e.g., from an HD-Zip protein); and (ii) amino acids 78to 181 of SEQ ID NO:11, amino acids 75 to 179 of SEQ ID NO:12, aminoacids 75 to 177 of SEQ ID NO:13, amino acids 76 to 186 of SEQ ID NO:15,or an amino acid sequence that is substantially similar or identicalthereto, wherein the HaHB11 polypeptide optionally binds CAAT(A/T)ATTG(SEQ ID NO:7). In embodiments of the invention, one, two three, four orall five of the leucines in the leucine zipper region are conserved.Optionally, the HaHB11 polypeptide further comprises amino acids 1 to 14of SEQ ID NO:3 or an amino acid sequence that is substantially similaror identical thereto. Alternatively, the HaHB11 polypeptides optionallyfurther comprises amino acids 1 to 14 of SEQ ID NO:14 or SEQ ID NO:15,or an amino acid sequence that is substantially similar or identicalthereto. In embodiments of the invention, the HaHB11 polypeptide isbiologically active.

In other representative embodiments, the leucine zipper region (e.g.,amino acids 76 to 110 of SEQ ID NO:3 or an equivalent thereof) isreplaced with a heterologous leucine zipper. For example, the HaHB11polypeptide can comprise (i) amino acids 15 to 74 of SEQ ID NO:3 or anamino acid sequence that is substantially similar or identical thereto;(ii) a leucine zipper domain (e.g., from an HD-Zip protein); and (iii)amino acids 111 to 175 of SEQ ID NO:3 or an amino acid sequence that issubstantially similar or identical thereto, wherein the HaHB11polypeptide optionally binds CAAT(A/T)ATTG (SEQ ID NO:7). In anotherexample, the HaHB11 polypeptide can comprise (i) amino acids 15 to 77 ofSEQ ID NO:10 or an amino acid sequence that is substantially similar oridentical thereto; (ii) a leucine zipper domain (e.g., from an HD-Zipprotein); and (iii) amino acids 116 to 180 of SEQ ID NO:10 or an aminoacid sequence that is substantially similar or identical thereto,wherein the HaHB11 polypeptide optionally binds CAAT(A/T)ATTG (SEQ IDNO:7). Optionally, the HaHB11 polypeptide further comprises amino acids1 to 14 of SEQ ID NO:3 or an amino acid sequence that is substantiallysimilar or identical thereto. In embodiments of the invention, theHaHB11 polypeptide is biologically active.

In other representative embodiments, the leucine zipper region ofproteins having the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15 (i.e., amino acids 78-113 ofSEQ ID NO:11, amino acids 75-110 of SEQ ID NO:12, amino acids 75-110 ofSEQ ID NO:13, amino acids 77 to 112 of SEQ ID NO:14 or amino acids 76 to111 of SEQ ID NO:15 or an equivalent thereof) is replaced with aheterologous leucine zipper.

In additional embodiments, the N-terminal region (e.g., amino acids 1 to14 of SEQ ID NO:3 or an equivalent thereof) is replaced with aheterologous N-terminal region from a HD-Zip protein. For example, theHaHB11 polypeptide can comprise (i) an N-terminal domain from an HD-Zipprotein; and (ii) amino acids 15 to 175 of SEQ ID NO:3 or an amino acidsequence that is substantially similar or identical thereto, wherein theHaHB11 polypeptide optionally binds CAAT(A/T)ATTG (SEQ ID NO:7). Inanother example, the HaHB11 polypeptide can comprise (i) an N-terminaldomain from an HD-Zip protein; and (ii) amino acids 15 to 77 of SEQ IDNO:10 or an amino acid sequence that is substantially similar oridentical thereto, wherein the HaHB11 polypeptide optionally bindsCAAT(A/T)ATTG (SEQ ID NO:7). In another example, the HaHB11 polypeptidecan comprise (i) an N-terminal domain from an HD-Zip protein; and (ii)amino acids 15 to 74 of SEQ ID NO:12 or amino acids 15 to 74 of SEQ IDNO:13, or an amino acid sequence that is substantially similar oridentical thereto, wherein the HaHB11 polypeptide optionally bindsCAAT(A/T)ATTG (SEQ ID NO:7). In embodiments of the invention, the HaHB11polypeptide is biologically active.

In embodiments of the invention, the C-terminal region (e.g., aminoacids 111 to 175 of SEQ ID NO:3 or an equivalent thereof) is replacedwith a heterologous C-terminal region from a HD-Zip protein. Forexample, the HaHB11 polypeptide can comprise: (i) amino acids 1 to 110of SEQ ID NO:3 or an amino acid sequence that is substantially similaror identical thereto; and (ii) a C-terminal domain from an HD-Zipprotein, wherein the HaHB11 polypeptide optionally binds CAAT(A/T)ATTG(SEQ ID NO:7). In another example, the HaHB11 polypeptide can comprise:(i) amino acids 1 to 113 of SEQ ID NO:10 or an amino acid sequence thatis substantially similar or identical thereto; and (ii) a C-terminaldomain from an HD-Zip protein, wherein the HaHB11 polypeptide optionallybinds CAAT(A/T)ATTG (SEQ ID NO:7). In another example, the HaHB11polypeptide can comprise: (i) amino acids 1 to 113 of SEQ ID NO:11, 1 to110 of SEQ ID NO:12; 1 to 110 of SEQ ID NO:13, 1 to 112 of SEQ ID NO:14,1 to 111 of SEQ ID NO:15, or an amino acid sequence that issubstantially similar or identical thereto; and (ii) a C-terminal domainfrom an HD-Zip protein, wherein the HaHB11 polypeptide optionally bindsCAAT(A/T)ATTG (SEQ ID NO:7). In embodiments of the invention, the HaHB11polypeptide is biologically active.

The invention also provides nucleic acids comprising one or more HaHB11expression control elements. In representative embodiments, the nucleicacid comprises, consists essentially of, or consists of a HaHB11promoter of the invention (including fragments thereof). The term“HaHB11 promoter” is intended to encompass a full-length promoter, andequivalents thereof (optionally, a biologically active equivalent) thathave substantially identical nucleotide sequences to the HaHB11 promotersequences specifically disclosed herein, as well as fragments of afull-length HaHB11 promoter (optionally, a biologically active fragment)and equivalents thereof (optionally, a biologically active equivalent)that have substantially identical nucleotide sequences to a fragment ofHaHB11 promoter sequences specifically disclosed herein. The term“HaHB11 promoter” includes sequences from Helianthus annuus as well asorthologs from other plant species, including naturally occurringallelic variants, isoforms, splice variants, and the like, or can bepartially or completely synthetic.

Orthologs from other organisms, in particular other plants, can beroutinely identified using methods known in the art. For example, PCRand other amplification techniques and hybridization techniques can beused to identify such orthologs based on their sequence similarity tothe sequences set forth herein.

Biological activities associated with the HaHB11 promoter include,without limitation, the ability to control or regulate transcription ofan operably associated coding sequence. Another non-limiting biologicalactivity includes the ability to bind one or more transcription factorsand/or RNA polymerase II. Other biological activities include theability to be induced in the same manner as a wild-type HaHB11 promoter,e.g., the ability to be induced by abiotic stress and/or ABA.

Thus, in exemplary embodiments, the isolated nucleic acid comprises,consists essentially of, or consists of the nucleotide sequence of SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 or an equivalent of any of theforegoing (optionally, a biologically active equivalent).

In other exemplary embodiments, the isolated nucleic acid comprises,consists essentially of, or consists of the nucleotide sequence of SEQID NO:16 or an equivalent of any of the foregoing (optionally, abiologically active equivalent). In a particular embodiment, theisolated nucleic acid consists of the nucleotide sequence of SEQ IDNO:16.

Equivalents of the HaHB11 promoters of the invention encompasspolynucleotides having substantial nucleotide sequence identity with theHaHB11 promoter sequences specifically disclosed herein (e.g., SEQ IDNO:4, SEQ ID NO:5 or SEQ ID NO:6) or fragments thereof, for example atleast about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% or more,and are optionally biologically active. In representative embodiments,there is no sequence variability in the TATA box, CAAT box, LTREelement, DRE element and/or ABRE element (see, e.g., the schematic inFIG. 1A), i.e., these sequences are conserved and any sequencevariability falls outside these regions.

The HaHB11 promoters of the invention also include polynucleotides thathybridize to the complete complement of the HaHB11 promoter sequencesspecifically disclosed herein (e.g., SEQ ID NO:4, SEQ ID NO:5 or SEQ IDNO:6) or fragments thereof under stringent hybridization conditions asknown by those skilled in the art and are optionally biologicallyactive.

In exemplary embodiments, the isolated nucleic acid comprises, consistsessentially of, or consists of the nucleotide sequence of SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6

In other embodiments, the HaHB11 promoters of the invention encompassportions of the endogenous HaHB11 promoter having substantial nucleotidesequence identity with the HaHB11 promoter having the sequence of SEQ IDNO:16, fragments thereof, for example at least about 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% or more, and are optionally biologicallyactive. In specific embodiments the HaHB11 promoters of the inventionencompass polynucleotides having substantial nucleotide sequenceidentity with nucleotides 1 to 800 or 1 to 500 of the HaHB11 longpromoter (SEQ ID NO:16), or fragments thereof of at least 50, 100 or 150nucleotides in length, for example at least about 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 99% or more, and are optionally biologicallyactive. In representative embodiments, there is no sequence variabilityin the LTRE element, DRE element and/or ABRE element.

The HaHB11 promoters of the invention also include portions of theendogenous HaHB11 promoter that hybridize to the complete complement ofnucleotides 1 to 800 or 1 to 500 of the HaHB11 long promoter (SEQ IDNO:16). The HaHB11 promoters of the invention also include portions ofthe endogenous HaHB11 promoter that hybridize to the complete complementof fragments of the HaHB11 long promoter (SEQ ID NO:16) of at least 50,100 or 150 nucleotides in length under stringent hybridizationconditions In particular embodiments, the HaHB11 promoters of theinvention hybridize to nucleotides 250 to 280 of SEQ ID NO:16 understringent hybridization conditions.

The HaHB11 promoter sequences encompass fragments (optionally,biologically active fragments) of the HaHB11 promoter sequencesspecifically disclosed herein (e.g., SEQ ID NO:4, SEQ ID NO:5 or SEQ IDNO:6) and equivalents thereof. Illustrative fragments comprise at leastabout 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,375, 400 or more nucleotides (optionally, contiguous nucleotides) of thefull-length sequence.

The HaHB11 promoter sequences encompass fragments (optionally,biologically active fragments) of the HaHB11 long promoter sequencedisclosed herein (SEQ ID NO:16) and equivalents thereof. Illustrativefragments comprise at least about 50, 60, 75, 100, 125, 150, 175, 200,225, 250, 275, 300, 325, 350, 375, 400 or more nucleotides (optionally,contiguous nucleotides) of nucleotides 1 to 800 of SEQ ID NO:16). Inparticular embodiments, the promoter sequences encompass nucleotides 1to 200, 200 to 400, 400 to 600, or 600 to 800 of SEQ ID NO:16 andcomprise at least about 225, 250, 275, 300, 325, 350, 375, 400, 500,700, 1,000, 1,250 or more nucleotides (optionally, contiguousnucleotides) of the sequence of SEQ ID NO:16.

In representative embodiments, the HaHB11 promoter sequence comprisesthe TATA box sequence, the CAAT box sequence, the LTRE (Low TemperatureResponsive Element; located at position 259 of SEQ ID NO:4 (−290 bp fromthe Transcription initiation Site (−strand)), the DRE (DehydrationResponsive Element; located at position 259 of SEQ ID NO:4 (−strand),and/or the ABRE (ABA Responsive Element; located at −266 of SEQ ID NO:4(−strand).

In representative embodiments, the HaHB11 promoter sequence comprisesthe LTRE sequence at position 1158 of SEQ ID NO:16, the ABRE-likesequence at position 1165 bp of SEQ ID NO:16; the ABRE-related sequencesat positions 864 and 1164 of SEQ ID NO:16; the DRE core at position 1158of SEQ ID NO:16, and/or the ANAERO2CONSENSUS at position 243 of SEQ IDNO:16).

In embodiments of the invention, the nucleic acid comprising the HaHB11promoter does not include any of the HaHB11 coding sequence.

Accordingly, in representative embodiments, the invention provides anucleic acid (e.g., a recombinant or isolated nucleic acid) comprising,consisting essentially of, or consisting of a nucleotide sequenceselected from the group consisting of: (a) SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6; (b) a nucleotide sequence comprising at least about 50, 60,75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 ormore nucleotides (optionally, contiguous nucleotides) of SEQ ID NO:4,SEQ ID NO:5 or SEQ ID NO:6; (c) a nucleotide sequence that hybridizes tothe complete complement of the nucleotide sequence of (a) or (b) understringent hybridization conditions; and (d) a nucleotide sequence havingat least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequenceidentity to the nucleotide sequences of any of (a) to (c). Inrepresentative embodiments, the nucleotide sequence is a biologicallyactive promoter sequence (e.g., has promoter activity) and is optionallyinduced by abiotic stress.

Accordingly, in representative embodiments, the invention provides anucleic acid (e.g., a recombinant or isolated nucleic acid) comprising,consisting essentially of the nucleotide sequence of: (a) SEQ ID NO:16;(b) nucleotides 1 to 200, 200 to 400, 400 to 600, or 600 to 800 of SEQID NO:10 and comprise at least about 225, 250, 275, 300, 325, 350, 375,400 or more nucleotides (optionally, contiguous nucleotides) of thesequence of SEQ ID NO:16; (c) a nucleotide sequence that hybridizes tothe complete complement of the nucleotide sequence of (b) understringent hybridization conditions; and (d) a nucleotide sequence havingat least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequenceidentity to the nucleotide sequences of any of (a) to (c). Inrepresentative embodiments, the nucleotide sequence is a biologicallyactive promoter sequence (e.g., has promoter activity) and is optionallyinduced by abiotic stress.

In embodiments of the invention, the nucleotide sequence comprises,consists essentially of, or consists of the nucleotide sequence of SEQID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

In embodiments, the HaHB11 promoter sequence of the invention isoperably associated with a nucleotide sequence of interest, which isoptionally a heterologous nucleotide sequence of interest. According tothis embodiment, the HaHB11 controls or regulates expression (e.g.,transcription and, optionally, translation) of the nucleotide sequenceof interest.

The invention further provides expression cassettes. In general, theexpression cassettes of the invention are of two general types.

First, the invention provides an expression cassette comprising a HaHB11nucleic acid encoding a HaHB11 polypeptide of the invention operablyassociated with a promoter. In embodiments, the nucleic acid encodingthe HaHB11 polypeptide is operably associated with a HaHB11 promotersequence of the invention. In embodiments, the nucleic acid encoding theHaHB11 polypeptide is operably associated with a heterologous promoter.

The heterologous promoter can be any suitable promoter known in the art(including bacterial, yeast, fungal, insect, mammalian, and plantpromoters). In particular embodiments, the promoter is a promoter forexpression in plants. The selection of promoters useable with thepresent invention can be made among many different types of promoters.Thus, the choice of promoter depends upon several factors, including,but not limited to, cell- or tissue-specific expression, desiredexpression level, efficiency, inducibility and/or selectability. Forexample, where expression in a specific tissue or organ is desired inaddition to inducibility, a tissue-specific promoter can be used (e.g.,a root specific promoter). In contrast, where expression in response toa stimulus is desired a promoter inducible by other stimuli or chemicalscan be used. Where continuous expression is desired throughout the cellsof a plant, a constitutive promoter can be chosen.

Non-limiting examples of constitutive promoters include cestrum viruspromoter (cmp) (U.S. Pat. No. 7,166,770), an actin promoter (e.g., therice actin 1 promoter; Wang et al., Mol. Cell. Biol. 12:3399-3406(1992); as well as U.S. Pat. No. 5,641,876), Cauliflower Mosaic Virus(CaMV) 35S promoter (Odell et al., Nature 313:810-812 (1985)), CaMV 19Spromoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987)), an opinesynthetase promoter (e.g., nos, mas, ocs, etc.; (Ebert et al., PNAS84:5745-5749 (1987)), Adh promoter (Walker et al., PNAS 84:6624-6629(1987)), sucrose synthase promoter (Yang & Russell, PNAS 87:4144-4148(1990)), and a ubiquitin promoter.

In some embodiments, the expression cassettes of the invention canfurther comprise enhancer elements and/or tissue preferred elements incombination with the promoter. In some embodiments, the expressioncassette comprises a constitutive S35 promoter operably associated witha polynucleotide sequence encoding HaHB11 having the amino acid sequenceof SEQ ID NO:3. In some embodiments, the expression cassette comprises aconstitutive S35 promoter operably associated with a polynucleotidesequence encoding HaHB11 having the amino acid sequence of SEQ ID NO:10.In some embodiments, the expression cassette comprises a constitutiveS35 promoter operably associated with a polynucleotide sequence encodingHaHB11 having the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

In some embodiments, the heterologous promoter is a promoter forexpression in a monocot plant. In further embodiments the heterologouspromoter is selected from: ZmUbi1 (Ubiquitin), Act1 (Actin), OsTubA1,(Tubulin), OsCc1 (Cytochrome c), rubi3 (polyubiquitin), APX (ascorbateperoxidase), SCP1, PGD1 (phosphogluconate dehydrogenase), R1G1B (earlydrought induced protein) and EIFS (translation initiation factor).

In some embodiments, the heterologous promoter is a promoter forexpression in a dicot plant. In further embodiments the heterologouspromoter is a CsVMV (cassava vein mosaic virus) or ScBV (sugarcanebacilliform badnavirus) promoter. In other embodiments, the heterologouspromoter is an CaMV 35S promoter.

Some non-limiting examples of tissue-specific promoters useable with thepresent invention include those driving the expression of seed storageproteins (e.g., 13-conglycinin, cruciferin, napin phaseolin, etc.), zeinor oil body proteins (such as oleosin), or proteins involved in fattyacid biosynthesis (including acyl carrier protein, stearoyi-ACPdesaturase and fatty acid desaturases (fad 2-1)), and other nucleicacids expressed during embryo development (such as Bce4, see, e.g.,Kridl et al., Seed Sci. Res. 1:209-219 (1991); as well as EP Patent No.255378). Thus, the promoters associated with these tissue-specificnucleic acids can be used in the present invention.

Additional examples of tissue-specific promoters usable with the presentinvention include, but are not limited to, the root-specific promotersRCc3 (Jeong et al., Plant Physiol. 153:185-197 (2010)) and RB7 (U.S.Pat. No. 5,459,252), the lectin promoter (Lindstrom et al., Der. Genet.11:160-167 (1990); and Vodkin et al., Prog. Clin. Biol. Res. 138:87-98(1983)), corn alcohol dehydrogenase 1 promoter (Dennis et al., NucleicAcids Res. 12:3983-4000 (1984)), S-adenosyi-L-methionine synthetase(SAMS) (Vander Mijnsbrugge et al., Plant and Cell Physiology,37(8):1108-1115 (1996)), corn light harvesting complex promoter (Bansalet al., PNAS 89:3654-3658 (1992)), corn heat shock protein promoter(O'Dell et al., EMBO J. 5:451-458 (1985); and Rochester et al., EMBO J.5:451-458 (1986)), pea small subunit RuBP carboxylase promoter(Cashmore, “Nuclear genes encoding the small subunit ofribulose-1,5-bisphosphate carboxylase” pp. 29-39 In: Genetic Engineeringof Plants, Hollaender ed., Plenum Press 1983; and Poulsen et al., Mol.Gen. Genet. 205:193-200 (1986)), Ti plasmid mannopine synthase promoter(Langridge et al., PNAS 86:3219-3223 (1989)), Ti plasmid nopalinesynthase promoter (Langridge et al., (1989), supra), petunia chalconeisomerase promoter (van Tunen et al., EMBO J. 7:1257-1263 (1988)), beanglycine rich protein 1 promoter (Keller et al., Genes Dev. 3:1639-1646(1989)), truncated CaMV 35S promoter (O'Dell et al., Nature 313:810-812(1985)), potato patatin promoter (Wenzler et al., Plant Mol. Biol.13:347-354 (1989)), root cell promoter (Yamamoto et al., Nucleic AcidsRes. 18:7449 (1990)), maize zein promoter (Kriz et al., Mol. Gen. Genet.207:90-98 (1987); Langridge et al., Cell 34:1015-1022 (1983); Reina etal., Nucleic Acids Res. 18:6425 (1990); Reina et al., Nucleic Acids Res.18:7449 (1990); and Wandelt et al., Nucleic Acids Res. 17:2354 (1989)),globulin-1 promoter (Belanger et al., Genetics 129:863-872 (1991)),a-tubulin cab promoter (Sullivan et al., Mol. Gen. Genet. 215:431-440(1989)), PEPCase promoter (Hudspeth et al., Plant Mol. Biol. 12:579-589(1989)), R gene complex-associated promoters (Chandler et al., PlantCell 1:1175-1183 (1989)), and chalcone synthase promoters (Franken etal., EMBO J. 10:2605-2612 (1991)). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., Mol. Gen. Genet.235:33-40 (1992); as well as U.S. Pat. No. 5,625,136). Other usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al., Science 270:1986-1988 (1995).

In addition, promoters functional in plastids can be used. Non-limitingexamples of such promoters include the bacteriophage T3 gene 9 5′ UTRand other promoters disclosed in U.S. Pat. No. 7,579,516. Otherpromoters useful with the present invention include but are not limitedto the S-E9 small subunit RuBP carboxylase promoter and the Kunitztrypsin inhibitor gene promoter (Kti3).

In some embodiments, inducible promoters can be used with the presentinvention. Examples of inducible promoters useable with the presentinvention include, but are not limited to, tetracycline repressor systempromoters, Lac repressor system promoters, copper-inducible systempromoters, salicylate-inducible system promoters (e.g., the PR1asystem), glucocorticoid-inducible promoters (Aoyama et al., Plant J.11:605-612 (1997)), and ecdysone-inducible system promoters. Othernon-limiting examples of inducible promoters include ABA- andturgor-inducible promoters, the auxin-binding protein gene promoter(Schwab et al., Plant J. 4:423-432 (1993)), the UDP glucose flavonoidglycosyl-transferase promoter (Ralston et al., Genetics 119:185-197(1988)), the 1VIPI proteinase inhibitor promoter (Cordero et al., PlantJ. 6:141-150 (1994)), the glyceraldehyde-3-phosphate dehydrogenasepromoter (Kohler et al., Plant Mol. Biol. 29:1293-1298 (1995); Martinezet al., J. Mol. Biol. 208:551-565 (1989); and Quigley et al., J. Mol.Evol. 29:412-421 (1989)) the benzene sulphonamide-inducible promoters(U.S. Pat. No. 5,364,780) and the glutathione S-transferase promoters.Likewise, one can use any appropriate inducible promoter described inGatz et al., Current Opinion Biotechnol. 7:168-172 (1996) and Gatz etal., Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108 (1997).

Other suitable promoters include promoters from viruses that infect thehost plant including, but not limited to, promoters isolated fromDasheen mosaic virus, Chlorella virus (e.g., the Chlorella virus adeninemethyltransferase promoter; Mitra et al., Plant Molecular Biology 26:85(1994)), tomato spotted wilt virus, tobacco rattle virus, tobacconecrosis virus, tobacco ring spot virus, tomato ring spot virus,cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, and thelike.

The invention also provides an expression cassette comprising a HaHB11promoter sequence of the invention, optionally in operable associationwith a nucleotide sequence of interest. The expression cassette canfurther have a plurality of restriction sites for insertion of anucleotide sequence of interest to be operably linked to the regulatoryregions. In embodiments, the HaHB11 promoter is operably associated witha nucleic acid encoding a HaHB11 polypeptide of the invention. Inembodiments, the HaHB11 promoter is operably associated with aheterologous nucleotide sequence of interest. In embodiments, the HaHB11promoter is operably associated with a heterologous nucleotide sequenceof interest. In embodiments, the HaHB11 promoter consists of thepolynucleotide ProH11 short sequence of SEQ ID NO:5. In furtherembodiments, the ProH11 short promoter (SEQ ID NO:5) is operablyassociated with a polynucleotide sequence encoding HaHB11.1 (SEQ IDNO:3) or HaHB11.1 (SEQ ID NO:10). In embodiments, the HaHB11 promoterconsists of the polynucleotide ProH11 long sequence of SEQ ID NO:16. Inadditional embodiments, the ProH11 long promoter (SEQ ID NO:16) isoperably associated with a polynucleotide sequence encoding HaHB11.1(SEQ ID NO:3) or HaHB11.1 (SEQ ID NO:10). In particular embodiments, theexpression cassette comprises more than one (e.g., two, three, four ormore) heterologous nucleotide sequences.

The expression cassettes of the invention may further comprise atranscriptional termination sequence. Any suitable termination sequenceknown in the art may be used in accordance with the present invention.The termination region may be native with the transcriptional initiationregion, may be native with the nucleotide sequence of interest, or maybe derived from another source. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthetase and nopaline synthetase termination regions. See also,Guerineau et al., Mol. Gen. Genet. 262:141 (1991); Proudfoot, Cell64:671 (1991); Sanfacon et al., Genes Dev. 5:141 (1991); Mogen et al.,Plant Cell 2:1261 (1990); Munroe et al., Gene 91:151 (1990); Ballas etal., Nucleic Acids Res. 17:7891 (1989); and Joshi et al., Nucleic AcidsRes. 15:9627 (1987). Additional exemplary termination sequences are thepea RubP carboxylase small subunit termination sequence and theCauliflower Mosaic Virus 35S termination sequence. Other suitabletermination sequences will be apparent to those skilled in the art.

Further, in particular embodiments, the nucleotide sequence of interest(e.g., heterologous nucleotide sequence of interest) is operablyassociated with a translational start site. The translational start sitecan be derived from the HaHB11 coding sequence or, alternatively, can bethe native translational start site associated with a heterologousnucleotide sequence of interest, or any other suitable translationalstart codon.

In illustrative embodiments, the expression cassette includes in the 5′to 3′ direction of transcription, a promoter, a nucleotide sequence ofinterest (e.g., a heterologous nucleotide sequence of interest), and atranscriptional and translational termination region functional inplants.

Those skilled in the art will understand that the expression cassettesof the invention can further comprise enhancer elements and/or tissuepreferred elements in combination with the promoter. In someembodiments, the expression cassette comprises a promoter sequenceoperably associated with the first intron of the Arabidopsis Cox5c2 (SEQID NO: 17). In some embodiments, the Cox5c2 is operably associated withthe long HaHB11 promoter (proH11 long; SEQ ID NO: 16). In embodiments,the Cox5c2 is operably associated with the short HaHB11 promoter (proH11short; SEQ ID NO: 5). In further embodiments, the proH11 short promoterand Cox5c2 sequence are operably associated with a polynucleotidesequence encoding HaHB11 having the amino acid sequence of SEQ ID NO:3or SEQ ID NO:10. In some embodiments, the Cox5c2 is operably associatedwith the long HaHB11 promoter (proH11 long; SEQ ID NO: 16).). In furtherembodiments, the proH11 long promoter and Cox5c2 sequence are operablyassociated with a polynucleotide sequence encoding HaHB11 having theamino acid sequence of SEQ ID NO:3 or SEQ ID NO:10.

Further, in some embodiments, it is advantageous for the expressioncassette to comprise a selectable marker gene for the selection oftransformed cells.

Selectable marker genes include genes encoding antibiotic resistance,such as those encoding neomycin phosphotransferase II (NEO) andhygromycin phosphotransferase (HP7), as well as genes conferringresistance to herbicidal compounds. Herbicide resistance genes generallycode for a modified target protein insensitive to the herbicide or foran enzyme that degrades or detoxifies the herbicide in the plant beforeit can act. See, DeBlock et al., EMBO J. 6:2513 (1987); DeBlock et al.,Plant Physiol. 91:691 (1989); Fromm et al., BioTechnology 8:833 (1990);Gordon-Kamm et al., Plant Cell 2:603 (1990). For example, resistance toglyphosphate or sulfonylurea herbicides has been obtained using genescoding for the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) and acetolactate synthase (ALS). Resistance toglufosinate ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate(2,4-D) have been obtained by using bacterial genes encodingphosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

Selectable marker genes that can be used according to the presentinvention further include, but are not limited to, genes encoding:neomycin phosphotransferase II (Fraley et al., CRC Critical Reviews inPlant Science 4:1 (1986)); cyanamide hydratase (Maier-Greiner et al.,PNAS 88:4250 (1991)); aspartate kinase; dihydrodipicolinate synthase(Peri et al., BioTechnology 11:715 (1993)); the bar gene (Toki et al.,Plant Physiol. 100:1503 (1992); Meagher et al., Crop Sci. 36:1367(1996)); tryptophane decarboxylase (Goddijn et al., Plant Mol. Biol.22:907 (1993)); neomycin phosphotransferase (NEO; Southern et al., J.Mol. Appl. Gen. 1:327 (1982)); hygromycin phosphotransferase (HPT orHYG; Shimizu et al., Mol. Cell. Biol. 6:1074 (1986)); dihydrofolatereductase (DHFR; Kwok et al., PNAS 83:4552 (1986)); phosphinothricinacetyltransferase (DeBlock et al., EMBO J. 6:2513 (1987));2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al., J.Cell. Biochem. 13D, 330 (1989)); acetohydroxyacid synthase (U.S. Pat.No. 4,761,373 to Anderson et al.; Haughn et al., Mol. Gen. Genet.221:266 (1988)); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comaiet al., Nature 317:741 (1985)); haloarylnitrilase (WO 87/04181 toStalker et al.); acetyl-coenzyme A carboxylase (Parker et al., PlantPhysiol. 92:1220 (1990)); dihydropteroate synthase (sutl; Guerineau etal., Plant Mol. Biol. 15:127 (1990)); and 32 kDa photosystem IIpolypeptide (psbA; Hirschberg et al., Science 222:1346 (1983)).

Also included are genes encoding resistance to: chloramphenicol(Herrera-Estrella et al., EMBO J. 2:987 (1983)); methotrexate(Herrera-Estrella et al., Nature 303:209 (1983); Meijer et al., PlantMol. Biol. 16:807 (1991)); hygromycin (Waldron et al., Plant Mol. Biol.5:103 (1985); Zhijian et al., Plant Science 108:219 (1995); Meijer etal., Plant Mol. Bio. 16:807 (1991)); streptomycin (Jones et al., Mol.Gen. Genet. 210:86 (1987)); and spectinomycin (Bretagne-Sagnard et al.,Transgenic Res. 5:131 (1996)); bleomycin (Hille et al., Plant Mol. Biol.7:171 (1986)); sulfonamide (Guerineau et al., Plant Mol. Bio. 15:127(1990); bromoxynil (Stalker et al., Science 242:419 (1988)); 2,4-D(Streber et al., Bio/Technology 7:811 (1989)); phosphinothricin (DeBlocket al., EMBO J. 6:2513 (1987)); spectinomycin (Bretagne-Sagnard et al.,Transgenic Research 5:131 (1996)).

Other selectable marker genes include the pat gene (for bialaphos andphosphinothricin resistance), the ALS gene for imidazolinone resistance,the HPH or HYG gene for hygromycin resistance, the Hm1 gene forresistance to the He-toxin, and other selective agents used routinelyand known to one of ordinary skill in the art. See generally, Yarranton,Curr. Opin. Biotech. 3:506 (1992); Chistopherson et al., PNAS 89: 6314(1992); Yao et al., Cell 71:63 (1992); Reznikoff, Mol. Microbial. 6:2419(1992); Barkley et al., THE OPERON 177-220 (1980); Hu et al., Cell48:555 (1987); Brown et al., Cell 49:603 (1987); Figge et al., Cell52:713 (1988); Deuschle et al., PNAS 86:5400 (1989); Fuerst et al., PNAS86:2549 (1989); Deuschle et al., Science 248:480 (1990); Labow et al.,Mol. Cell. Biol. 10:3343 (1990); Zambretti et al., PNAS 89:3952 (1992);Bairn et al., PNAS 88:5072 (1991); Wyborski et al., Nuc. Acids Res.19:4647 (1991); Hillenand-Wissman, Topics in Mol. and Struct. Biol.10:143 (1989); Degenkolb et al., Antimicrob. Agents Chemother. 35:1591(1991); Kleinschnidt et al., Biochemistry 27:1094 (1988); Gatz et al.,Plant J. 2:397 (1992); Gossen et al., PNAS 89:5547 (1992); Oliva et al.,Antimicrob. Agents Chemother. 36:913 (1992); HLAVKA A L., HANDBOOK OFEXPERIMENTAL PHARMACOLOGY 78 (1985); and Gill et al., Nature 334:721(1988).

The nucleotide sequence of interest can additionally be operably linkedto a sequence that encodes a transit peptide that directs expression ofan encoded polypeptide of interest to a particular cellular compartment.Transit peptides that target protein accumulation in higher plant cellsto the chloroplast, mitochondrion, vacuole, nucleus, and the endoplasmicreticulum (for secretion outside of the cell) are known in the art.Transit peptides that target proteins to the endoplasmic reticulum aredesirable for correct processing of secreted proteins. Targeting proteinexpression to the chloroplast (for example, using the transit peptidefrom the RubP carboxylase small subunit gene) has been shown to resultin the accumulation of very high concentrations of recombinant proteinin this organelle. The pea RubP carboxylase small subunit transitpeptide sequence has been used to express and target mammalian genes inplants (U.S. Pat. Nos. 5,717,084 and 5,728,925 to Herrera-Estrella etal.). Alternatively, mammalian transit peptides can be used to targetrecombinant protein expression, for example, to the mitochondrion andendoplasmic reticulum. It has been demonstrated that plant cellsrecognize mammalian transit peptides that target endoplasmic reticulum(U.S. Pat. Nos. 5,202,422 and 5,639,947 to Hiatt et al.).

Further, the expression cassette can comprise a 5′ leader sequence thatacts to enhance expression (transcription, post-transcriptionalprocessing and/or translation) of an operably associated nucleotidesequence of interest. Leader sequences are known in the art and includesequences from: picornavirus leaders, e.g., EMCV leader(Encephalomyocarditis 5′ noncoding region; Elroy-Stein et al., PNASUSA,86:6126 (1989)); potyvirus leaders, e.g., TEV leader (Tobacco EtchVirus; Allison et al., Virology, 154:9 (1986)); human immunoglobulinheavy-chain binding protein (BiP; Macajak and Sarnow, Nature 353:90(1991)); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4; Jobling and Gehrke, Nature 325: 622 (1987));tobacco mosaic virus leader (TMV; Gallie, MOLECULAR BIOLOGY OF RNA,237-56 (1989)); and maize chlorotic mottle virus leader (MCMV; Lommel etal., Virology 81:382 (1991)). See also, Della-Cioppa et al., PlantPhysiology 84:965 (1987).

The heterologous nucleotide sequence(s) in the expression cassette canbe any nucleotide sequence(s) of interest and can be obtained fromprokaryotes or eukaryotes (e.g., bacteria, fungi, yeast, viruses,plants, mammals) or the heterologous nucleotide sequence can besynthesized in whole or in part. Further, the heterologous nucleotidesequence can encode a polypeptide or can be transcribed to produce afunctional RNA. In particular embodiments, the functional RNA can beexpressed to improve an agronomic trait in the plant (e.g., droughtresistance, heat resistance, salt resistance, disease resistance, insectand other pest resistance [e.g., a Bacillus thuringiensis endotoxin],herbicide resistance, and the like), to confer male sterility, toimprove fertility and/or enhance nutritional quality (e.g., enzymes thatenhance nutritional quality). The nucleotide sequence may further beused in the sense orientation to achieve suppression of endogenous plantgenes, as is known by those skilled in the art (see, e.g., U.S. Pat.Nos. 5,283,184; and 5,034,323).

The heterologous nucleotide sequence can encode a polypeptide thatimparts a desirable agronomic trait to the plant (as described above),confers male sterility, improves fertility and/or improves nutritionalquality. Other suitable polypeptides include enzymes that can degradeorganic pollutants or remove heavy metals. Such plants, and the enzymesthat can be isolated therefrom, are useful in methods of environmentalprotection and remediation. Alternatively, the heterologous nucleotidesequence can encode a therapeutically or pharmaceutically usefulpolypeptide or an industrial polypeptide (e.g., an industrial enzyme).Such polypeptides include, but are not limited to antibodies andantibody fragments, cytokines, hormones, growth factors, receptors,enzymes and the like.

Heterologous nucleotide sequences suitable to confer tolerance to theherbicide glyphosate include, but are not limited to the Agrobacteriumstrain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described inU.S. Pat. No. 5,633,435 or the glyphosate oxidoreductase gene (GOX) asdescribed in U.S. Pat. No. 5,463,175. Other heterologous nucleotidesequences include genes conferring resistance to herbicides that act toinhibit the action of acetolactate synthase (ALS), in particular thesulfonylurea-type herbicides (e.g., mutant forms of the acetolactatesynthase (ALS) gene that lead to such resistance, in particular the S4and/or Hra mutations), genes coding for resistance to herbicides thatact to inhibit the action of glutamine synthase, such asphosphinothricin or basta (e.g., the bar gene). The bar gene encodesresistance to the herbicide basta, the nptll gene encodes resistance tothe antibiotics kanamycin and geneticin, and the ALS gene encodesresistance to the herbicide chlorsulfuron.

Suitable heterologous nucleotide sequences that confer insect toleranceinclude those which provide resistance to pests such as rootworm,cutworm, European Corn Borer, and the like. Exemplary nucleotidesequences include, but are not limited to, a Bacillus insect controlprotein gene (see, e.g., WO 99/31248; U.S. Pat. Nos. 5,689,052;5,500,365; 5,880,275); Bacillus thuringiensis toxic protein genes (see,e.g., U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; 6,555,655; 6,541,448; and U.S. Pat. No. 6,538,109; Geiser, etal., Gene 48:109 (1986)); and lectins (Van Damme et al., Plant Mol.Biol. 24:825 (1994)).

Alternatively, the heterologous nucleotide sequence can encode areporter polypeptide (e.g., an enzyme), including but not limited toGreen Fluorescent Protein, beta-galactosidase, luciferase, alkalinephosphatase, the GUS gene encoding beta-glucuronidase, andchloramphenicol acetyltransferase.

Where appropriate, the heterologous nucleic acids may be optimized forincreased expression in a transformed plant, e.g., by using plantpreferred codons. Methods for synthetic optimization of nucleic acidsequences are available in the art. The nucleotide sequence can beoptimized for expression in a particular host plant or alternatively canbe modified for optimal expression in monocots. See, e.g., EP 0 359 472,EP 0 385 962, WO 91/16432; Perlak et al., PNAS 88, 3324 (1991), andMurray et al., Nucl. Acids Res. 17:477 (1989), and the like. Plantpreferred codons can be determined from the codons of highest frequencyin the proteins expressed in that plant. Additional sequencemodifications are known to enhance gene expression in a cellular host.These include elimination of sequences encoding spurious polyadenylationsignals, exon-intron splice site signals, transposon-like repeats, andother such well-characterized sequences which may be deleterious to geneexpression. The G-C content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to knowngenes expressed in the host cell. When possible, the sequence ismodified to avoid predicted hairpin secondary mRNA structures.

The invention further provides vectors comprising the nucleic acids andexpression cassettes of the invention, including expression vectors,transformation vectors and vectors for replicating and/or manipulatingthe nucleotide sequences in the laboratory. The vector can be a plantvector, animal (e.g., insect or mammalian) vector, bacterial vector,yeast vector or fungal vector. Generally, according to the presentinvention, the vector is a plant vector, a bacterial vector, or ashuttle vector that can replicate in either host under appropriateconditions. Bacterial and plant vectors are well-known in the art.Exemplary plant vectors include plasmids (e.g., pUC or the Ti plasmid),cosmids, phage, bacterial artificial chromosomes (BACs), yeastartificial chromosomes (YACs) and plant viruses.

III. Transgenic Plants, Plant Parts and Plant Cells

The invention also provides transgenic plants, plant parts and plantcells comprising the nucleic acids, expression cassettes and vectors ofthe invention.

Accordingly, as one aspect the invention provides a cell comprising anucleic acid, expression cassette, or vector of the invention. The cellcan be transiently or stably transformed with the nucleic acid,expression cassette or vector. Further, the cell can be a cultured cell,a cell obtained from a plant, plant part, or plant tissue, or a cell insitu in a plant, plant part or plant tissue. Cells can be from anysuitable species, including plant (e.g., Helianthus annuus), bacterial,yeast, insect and/or mammalian cells. In representative embodiments, thecell is a plant cell or bacterial cell.

The invention also provides a plant part (including a plant tissueculture) comprising a nucleic acid, expression cassette, or vector ofthe invention. The plant part can be transiently or stably transformedwith the nucleic acid, expression cassette or vector. Further, the plantpart can be in culture, can be a plant part obtained from a plant, or aplant part in situ. In representative embodiments, the plant partcomprises a cell of the invention, as described in the precedingparagraph.

Seed comprising the nucleic acid, expression cassette, or vector of theinvention are also provided. Optionally, the nucleic acid, expressioncassette or vector is stably incorporated into the genome of the seed.

The invention also contemplates a transgenic plant comprising a nucleicacid, expression cassette, or vector of the invention. The plant can betransiently or stably transformed with the nucleic acid, expressioncassette or vector. In representative embodiments, the plant comprises acell or plant part of the invention, as described in the precedingparagraphs. In representative embodiments, wherein the nucleic acid,expression cassette or vector encodes a HaHB11 polypeptide of theinvention, the transgenic plant has increased tolerance to an abioticstress, increased yield (e.g., under normal cultivation conditionsand/or conditions of mild and/or severe abiotic stress), increasedmature height and/or delayed development and/or a prolonged life span.In representative embodiments, wherein the nucleic acid, expressioncassette or vector comprises a HaHB11 promoter of the invention, theplant expresses a nucleotide sequence of interest (e.g., a heterologousnucleotide sequence of interest) operably associated with the HaHB11promoter, where expression is optionally induced by an abiotic stress.

In a representative embodiment, a transgenic plant is stably transformedwith an isolated nucleic acid encoding a polypeptide selected from thegroup consisting of: (a) a polypeptide comprising (i) a HD-Zip domainthat binds CAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 15 to 113 ofSEQ ID NO:10; and (b) a polypeptide comprising (i) a HD-Zip domain thatbinds CAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence thatis at least about 96% identical to amino acids 15 to 181 of SEQ IDNO:10. In some embodiments, the transgenic plant is stably transformedwith an isolated nucleic acid encoding the HAHB11 polypeptide of SEQ IDNO:3. In some embodiments, the transgenic plant is stably transformedwith an isolated nucleic acid encoding the HAHB11 polypeptide of SEQ IDNO:10.

In further embodiments, the plant is stably transformed with a nucleicacid sequence comprising the nucleotide sequence of SEQ ID NO:9. In someembodiments the transgenic plant is stably transformed with anexpression cassette comprising the isolated nucleic acid operablyassociated with a promoter. In further embodiments, the expressioncassette comprises a promoter containing the nucleotide sequence of SEQID NO:5 or SEQ ID NO:16. In additional embodiments, the expressioncassette comprises a heterologous promoter. In additional embodiments,the expression cassette comprises a selectable marker.

Still further, the invention encompasses a crop comprising a pluralityof the transgenic plants of the invention, as described herein.Nonlimiting examples of the types of crops comprising a plurality oftransgenic plants of the invention include an agricultural field, a golfcourse, a residential lawn or garden, a public lawn or garden, a roadside planting, an orchard, and/or a recreational field (e.g., acultivated area comprising a plurality of the transgenic plants of theinvention).

Products harvested from the plants of the invention are also provided.Nonlimiting examples of a harvested product include a seed (e.g.,sunflower seeds), a leaf, a stem, a shoot, a fruit, flower, root,biomass (e.g., for biofuel production) and/or extract.

In some embodiments, a processed product produced from the harvestedproduct is provided. Nonlimiting examples of a processed product includea protein (e.g., a recombinant protein), an extract, a medicinal product(e.g., artemicin as an antimalarial agent), a fiber or woven textile, afragrance, dried fruit, a biofuel (e.g., ethanol), a tobacco product(e.g., cured tobacco, cigarettes, chewing tobacco, cigars, and thelike), an oil (e.g., sunflower oil, corn oil, canola oil, and the like),a nut butter, a seed butter (e.g., sunflower butter), a flour or meal(e.g., wheat flour, corn meal) and/or any other animal feed (e.g., soy,maize, barley, rice, alfalfa) and/or human food product (e.g., aprocessed wheat, maize, rice or soy food product). Further, processedproduct can be cut, dried, cooked, canned, frozen, dehydrated, powdered,ground and/or mixed with other ingredients.

IV. Methods of Introducing Nucleic Acids

The invention also provides methods of delivering a nucleic acid,expression cassette or vector of the invention to a target plant orplant cell (including callus cells or protoplasts), plant parts, seed,plant tissue (including callus), and the like. The invention furthercomprises host plants, cells, plant parts, seeds, tissue culture(including callus) transiently or stably transformed with the nucleicacids, expression cassettes or vectors of the invention.

The invention further provides a method of expressing a HaHB11polypeptide in a plant, plant part or plant cell. In representativeembodiments, the method comprises transforming the plant, plant part orplant cell with a nucleic acid, expression cassette, or vector of theinvention encoding the HaHB11 polypeptide. The plant can be transientlyor stably transformed. This method finds use, for example, in methods ofevaluating the structure and/or function of HaHB11.

The invention also encompasses a method of increasing tolerance of aplant to abiotic stress, the method comprising: (a) stably transforminga plant cell with a nucleic acid, expression cassette, or vectorencoding an HaHB11 polypeptide of the invention; (b) regenerating astably transformed plant from the stably transformed plant cell of (a);and (c) expressing the nucleotide sequence in the plant (e.g., in anamount effective to increase the tolerance of the plant to an abioticstress). The method optionally includes the further step of exposing theplant to the abiotic stress (e.g., growing the plant under the abioticstress conditions).

The invention further provides a method of increasing the yield from aplant, the method comprising: (a) stably transforming a plant cell witha nucleic acid, expression cassette, or vector encoding an HaHB11polypeptide of the invention; (b) regenerating a stably transformedplant from the stably transformed plant cell of (a); and (c) expressingthe nucleotide sequence in the plant (e.g., in an amount effective toincrease the yield from the plant). According to this embodiment, theyield of the plant can be increased under normal growth conditions(e.g., normal irrigation and salt conditions) and/or conditions of mildabiotic stress and/or severe abiotic stress. Still further, theinvention provides a method of the prolonging (e.g., increasing) thelife span and/or delaying development of a plant, the method comprising:(a) stably transforming a plant cell with a nucleic acid, expressioncassette, or vector encoding an HaHB11 polypeptide of the invention; (b)regenerating a stably transformed plant from the stably transformedplant cell of (a); and (c) expressing the nucleotide sequence in theplant (e.g., in an amount effective to prolong the life span and/ordelay the development of a plant). Without wishing to be bound by anytheory of the invention, it appears that the increase in yield seen uponectopic expression of HaHB11 may result, at least in part, from thedelayed development and prolonged life span of the transgenic plant. Inrepresentative embodiments, the plant is a turfgrass (including a foragegrass or ornamental grass), a biomass grass, or an ornamental plant.

Abiotic stress is as described elsewhere herein. In representativeembodiments of the foregoing methods, the abiotic stress comprisesdrought, salt stress, submergence stress and/or waterlogging stress,and/or stress after removal of a submergence stressor (e.g.,desubmergence stress).

In some embodiments, the methods of the invention can be used toinvention increase yield and/or increasing tolerance of a plant toabiotic stress. In further embodiments, the abiotic stress comprisesdrought, salt stress, waterlogging stress, submergence stress, and/ordesubmergence stress. Thus, in some embodiments, the transgenic plantsof the invention are grown under the abiotic stress conditions.Alternatively, in additional embodiments, the transgenic plants aregrown under normal cultivation conditions.

In one embodiment, the invention provides a method of increasing yieldand/or increasing tolerance of a plant to abiotic stress, the methodcomprising: (a) stably transforming a plant cell with an isolatednucleic acid encoding a polypeptide selected from the group consistingof: (i) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) amino acids 15 to 113 of SEQ IDNO:10; and (ii) a polypeptide comprising (i) a HD-Zip domain that bindsCAAT(A/T)ATTG (SEQ ID NO:7) and (ii) an amino acid sequence that is atleast about 96% identical to amino acids 15 to 181 of SEQ ID NO:10. In afurther embodiment, the invention further includes regenerating a stablytransformed plant from the stably transformed plant cell; and expressingthe nucleotide sequence in the plant. In some embodiments, the methodincludes stably transforming a plant cell with an isolated nucleic acidencoding the HAHB11 polypeptide of SEQ ID NO:3 or SEQ ID NO:10.

The invention also provides methods of introducing a nucleic acid into aplant, plant part or plant cell. In representative embodiments, themethod comprises transforming the plant, plant part or plant cell with anucleic acid, expression cassette, or vector comprising an HaHB11promoter of the invention, optionally, in operable association with anucleotide sequence of interest (e.g., a heterologous nucleotidesequence of interest). In embodiments, the nucleotide sequence ofinterest encodes an HaHB11 polypeptide of the invention (e.g., SEQ IDNO:1, nucleotides 7-944 of SEQ ID NO:1, SEQ ID NO:2). In embodiments,the nucleotide sequence of interest encodes an HaHB11 polypeptide havingthe amino acid sequence of SEQ ID NO:10 (e.g., SEQ ID NO:9). Inembodiments, the nucleotide sequence of interest encodes an HaHB11polypeptide having the amino acid sequence of SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. In embodiments of theinvention the promoter is operably associated with a nucleotide sequenceof interest that is heterologous to the promoter. The plant can betransiently or stably transformed. This method finds use, for example,in methods of evaluating the structure and/or function of the HaHB11promoter.

As another aspect, the invention provides a method of stably expressinga nucleotide sequence of interest in a plant. According torepresentative embodiments, the method comprises: (a) stablytransforming a plant cell with an expression cassette or vectorcomprising an HaHB11 promoter of the invention operably associated witha nucleotide sequence of interest (e.g., a heterologous nucleotidesequence of interest); (b) regenerating a stably transformed plant fromthe stably transformed plant cell of (a); and (c) expressing thenucleotide sequence of interest in the plant. In embodiments, thenucleotide sequence of interest encodes an HaHB11 polypeptide of theinvention (e.g., SEQ ID NO:1, nucleotides 7-944 of SEQ ID NO:1, SEQ IDNO:2). In embodiments, the nucleotide sequence of interest encodes anHaHB11 polypeptide having the amino acid sequence of SEQ ID NO:10 (e.g.,SEQ ID NO:9). In embodiments, the nucleotide sequence of interestencodes an HaHB11 polypeptide having the amino acid sequence of SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. Inembodiments of the invention the promoter is operably associated with anucleotide sequence of interest that is heterologous to the promoter,which may optionally encode a polypeptide (e.g., as described in moredetail elsewhere herein). The method can further comprise the step ofcollecting the polypeptide. Alternatively, in some embodiments, theheterologous nucleotide sequence of interest can be transcribed toproduce a functional RNA.

Also contemplated by the present invention is a method of producing apolypeptide in a plant, plant tissue culture or plant cell, wherein theplant, plant tissue culture, or plant cell comprises an expressioncassette or vector comprising an HaHB11 promoter of the inventionoperably associated with a nucleotide sequence of interest (e.g., aheterologous nucleotide sequence of interest, wherein the nucleotidesequence of interest encodes a polypeptide. In embodiments, thenucleotide sequence of interest encodes an HaHB11 polypeptide of theinvention (e.g., SEQ ID NO:1, nucleotides 7-944 of SEQ ID NO:1, SEQ IDNO:2). In embodiments, the nucleotide sequence of interest encodes anHaHB11 polypeptide having the amino acid sequence of SEQ ID NO:10 (e.g.,SEQ ID NO:9). In embodiments, the nucleotide sequence of interestencodes an HaHB11 polypeptide having the amino acid sequence of SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15. Inembodiments of the invention the promoter is operably associated with anucleotide sequence of interest that is heterologous to the promoter. Inrepresentative embodiments, the method comprises growing the plant orculturing the tissue culture or plant cell under conditions sufficientfor the production of the polypeptide in the plant, plant tissue cultureor plant cell and, optionally, collecting the polypeptide. According tothis embodiment, the polypeptide can be a secreted polypeptide.

The invention also encompasses transgenic plants, plant parts, and plantcells produced by the methods of the invention.

Also provided by the invention are seed produced from the inventivetransgenic plants. Optionally, the seed comprise a nucleic acid,expression cassette or vector of the invention stably incorporated intothe genome.

Methods of introducing nucleic acids, transiently or stably, intoplants, plant tissues, cells, protoplasts, seed, callus and the like areknown in the art. Stably transformed nucleic acids can be incorporatedinto the genome. Exemplary transformation methods include biologicalmethods using viruses and Agrobacterium, physicochemical methods such aselectroporation, floral dip methods, polyethylene glycol, ballisticbombardment, microinjection, and the like. Other transformationtechnology includes the whiskers technology that is based on mineralfibers (see •e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765) and pollentube transformation. In one form of direct transformation, the vector ismicroinjected directly into plant cells by use of micropipettes tomechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genetics202:179 (1985)).

In another protocol, the genetic material is transferred into the plantcell using polyethylene glycol (Krens et al., Nature 296:72 (1982)).

In still another method, protoplasts are fused with minicells, cells,lysosomes, or other fusible lipid-surfaced bodies that contain thenucleotide sequence to be transferred to the plant (Fraley et al., PNAS79:1859 (1982)).

Nucleic acids may also be introduced into the plant cells byelectroporation (Fromm et al., PNAS 82:5824 (1985)). In this technique,plant protoplasts are electroporated in the presence of nucleic acidscomprising the expression cassette. Electrical impulses of high fieldstrength reversibly permeabilize biomembranes allowing the introductionof the nucleic acid. Electroporated plant protoplasts reform the cellwall, divide and regenerate. One advantage of electroporation is thatlarge pieces of DNA, including artificial chromosomes, can betransformed by this method.

Ballistic transformation typically comprises the steps of: (a) providinga plant material as a target; (b) propelling a microprojectile carryingthe heterologous nucleotide sequence at the plant target at a velocitysufficient to pierce the walls of the cells within the target and todeposit the nucleotide sequence within a cell of the target to therebyprovide a transformed target. The method can further include the step ofculturing the transformed target with a selection agent and, optionally,regeneration of a transformed plant. As noted below, the technique maybe carried out with the nucleotide sequence as a precipitate (wet orfreeze-dried) alone, in place of the aqueous solution containing thenucleotide sequence.

Any ballistic cell transformation apparatus can be used in practicingthe present invention. Exemplary apparatus are disclosed by Sandford etal., Particulate Science and Technology 5:27 (1988)), Klein et al.,Nature 327:70 (1987)), and in EP 0 270 356. Such apparatus have beenused to transform maize cells (Klein et al., PNAS 85:4305 (1988)),soybean callus (Christou et al., Plant Physiol. 87:671 (1988)), McCabeet al., BioTechnology 6:923 (1988), yeast mitochondria (Johnston et al.,Science 240:1538 (1988)), and Chlamydomonas chloroplasts (Boynton etal., Science 240:1534 (1988)).

Alternately, an apparatus configured as described by Klein et al.,(Nature 70:327 (1987)) may be utilized. This apparatus comprises abombardment chamber, which is divided into two separate compartments byan adjustable-height stopping plate. An acceleration tube is mounted ontop of the bombardment chamber. A macroprojectile is propelled down theacceleration tube at the stopping plate by a gunpowder charge. Thestopping plate has a borehole formed therein, which is smaller indiameter than the microprojectile. The macroprojectile carries themicroprojectile(s), and the macroprojectile is aimed and fired at theborehole. When the macroprojectile is stopped by the stopping plate, themicroprojectile(s) is propelled through the borehole. The target ispositioned in the bombardment chamber so that a microprojectile(s)propelled through the bore hole penetrates the cell walls of the cellsin the target and deposit the nucleotide sequence of interest carriedthereon in the cells of the target. The bombardment chamber is partiallyevacuated prior to use to prevent atmospheric drag from unduly slowingthe microprojectiles. The chamber is only partially evacuated so thatthe target tissue is not desiccated during bombardment. A vacuum ofbetween about 400 to about 800 millimeters of mercury is suitable.

In alternate embodiments, ballistic transformation is achieved withoutuse of microprojectiles. For example, an aqueous solution containing thenucleotide sequence of interest as a precipitate may be carried by themacroprojectile (e.g., by placing the aqueous solution directly on theplate-contact end of the macroprojectile without a microprojectile,where it is held by surface tension), and the solution alone propelledat the plant tissue target (e.g., by propelling the macroprojectile downthe acceleration tube in the same manner as described above). Otherapproaches include placing the nucleic acid precipitate itself (“wet”precipitate) or a freeze-dried nucleotide precipitate directly on theplate-contact end of the macroprojectile without a microprojectile. Inthe absence of a microprojectile, it is believed that the nucleotidesequence must either be propelled at the tissue target at a greatervelocity than that needed if carried by a microprojectile, or thenucleotide sequenced caused to travel a shorter distance to the target(or both).

It particular embodiments, the nucleotide sequence fs delivered by amicroprojectile. The microprojectile can be formed from any materialhaving sufficient density and cohesiveness to be propelled through thecell wall, given the particle's velocity and the distance the particlemust travel. Non-limiting examples of materials for makingmicroprojectiles include metal, glass, silica, ice, polyethylene,polypropylene, polycarbonate, and carbon compounds (e.g., graphite,diamond). Non-limiting examples of suitable metals include tungsten,gold, and iridium. The particles should be of a size sufficiently smallto avoid excessive disruption of the cells they contact in the targettissue, and sufficiently large to provide the inertia required topenetrate to the cell of interest in the target tissue. Particlesranging in diameter from about one-half micrometer to about threemicrometers are suitable. Particles need not be spherical, as surfaceirregularities on the particles may enhance their carrying capacity.

The nucleotide sequence may be immobilized on the particle byprecipitation. The precise precipitation parameters employed will varydepending upon factors such as the particle acceleration procedureemployed, as is known in the art. The carrier particles may optionallybe coated with an encapsulating agents such as polylysine to improve thestability of nucleotide sequences immobilized thereon, as discussed inEP 0 270 356 (column 8).

Alternatively, plants may be transformed using Agrobacterium tumefaciensor Agrobacterium rhizogenes. Agrobacterium-mediated nucleic acidtransfer exploits the natural ability of A. tumefaciens and A.rhizogenes to transfer DNA into plant chromosomes. Agrobacterium is aplant pathogen that transfers a set of genes encoded in a region calledT-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, into plant cells. The typical result of transfer of the Tiplasmid is a tumorous growth called a crown gall in which the T-DNA isstably integrated into a host chromosome. Integration of the Ri plasmidinto the host chromosomal DNA results in a condition known as “hairyroot disease”. The ability to cause disease in the host plant can beremoved by deletion of the genes in the T-DNA without loss of DNAtransfer and integration. The DNA to be transferred is attached toborder sequences that define the end points of an integrated T-DNA.

Transfer by means of engineered Agrobacterium strains has become routinefor many dicotyledonous plants. Some difficulty has been experienced,however, in using Agrobacterium to transform monocotyledonous plants, inparticular, cereal plants. However, Agrobacterium mediatedtransformation has been achieved in several monocot species, includingcereal species such as rye, maize (Rhodes et al., Science 240, 204(1988)), and rice (Hiei et al., (1994) Plant J. 6:271).

While the following discussion will focus on using A. tumefaciens toachieve gene transfer in plants, those skilled in the art willappreciate that this discussion also applies to A. rhizogenes.Transformation using A. rhizogenes has developed analogously to that ofA. tumefaciens and has been successfully utilized to transform, forexample, alfalfa, Solanum nigrum L., and poplar (U.S. Pat. No. 5,777,200to Ryals et al.). As described by U.S. Pat. No. 5,773,693 to Burgess etal., it is preferable to use a disarmed A. tumefaciens strain (asdescribed below), however, the wild-type A. rhizogenes may be employed.An illustrative strain of A. rhizogenes is strain 15834.

In particular protocols, the Agrobacterium strain is modified to containthe nucleotide sequences to be transferred to the plant. The nucleotidesequence to be transferred is incorporated into the T-region and istypically flanked by at least one T-DNA border sequence, optionally twoT-DNA border sequences. A variety of Agrobacterium strains are known inthe art particularly, and can be used in the methods of the invention.See, e.g., Hooykaas, Plant Mol. Biol. 13:327 (1989); Smith et al., CropScience 35:301 (1995); Chilton, PNAS 90, 3119 (1993); Mollony et al.,Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al., NatureBiotechnol. 14:745 (1996); and Komari et al., The Plant J. 10:165(1996).

In addition to the T-region, the Ti (or Ri) plasmid contains a virregion. The vir region is important for efficient transformation, andappears to be species-specific.

Two exemplary classes of recombinant Ti and Ri plasmid vector systemsare commonly used in the art. In one class, called “cointegrate,” theshuttle vector containing the gene of interest is inserted by geneticrecombination into a non-oncogenic Ti plasmid that contains both thecis-acting and trans-acting elements required for plant transformationas, for example, in the PMLJ1 shuttle vector of DeBlock et al., EMBO J.3:1681 (1984), and the non-oncogenic Ti plasmid pGV2850 described byZambryski et al., EMBOJ 2:2143 (1983). In the second class or “binary”system, the gene of interest is inserted into a shuttle vectorcontaining the cis-acting elements required for plant transformation.The other necessary functions are provided in trans by the non-oncogenicTi plasmid as exemplified by the pBIN19 shuttle vector described byBevan, Nucleic Acids Research 12:8711 (1984), and the non-oncogenic Tiplasmid PAL4404 described by Hoekma, et al., Nature 303:179 (1983).

Binary vector systems have been developed where the manipulated disarmedT-DNA carrying the heterologous nucleotide sequence of interest and thevir functions are present on separate plasmids. In this manner, amodified T-DNA region comprising foreign DNA (the nucleic acid to betransferred) is constructed in a small plasmid that replicates in E.coli. This plasmid is transferred conjugatively in a tri-parental matingor via electroporation into A. tumefaciens that contains a compatibleplasmid with virulence gene sequences. The vir functions are supplied intrans to transfer the T-DNA into the plant genome. Such binary vectorsare useful in the practice of the present invention.

In particular embodiments of the invention, super-binary vectors areemployed. See, e.g., U.S. Pat. No. 5,591,615 and EP 0 604 662. Such asuper-binary vector has been constructed containing a DNA regionoriginating from the hypervirulence region of the Ti plasmid pTiBo542(Jin et al., J. Bacterial. 169:4417 (1987)) contained in asuper-virulent A. tumefaciens A281 exhibiting extremely hightransformation efficiency (Hood et al., Biotechnol. 2:702 (1984); Hoodet al., J. Bacterial. 168:1283 (1986); Komari et al., J. Bacterial.166:88 (1986); Jin et al., J. Bacterial. 169:4417 (1987); Komari, PlantScience 60:223 (1987); ATCC Accession No. 37394.

Exemplary super-binary vectors known to those skilled in the art includepTOK162 (Japanese patent Appl. (Kokai) No. 4-222527, EP 504,869, EP604,662, and U.S. Pat. No. 5,591,616) and pTOK233 (Komari, Plant CellReports 9:303 (1990); Ishida et al., Nature Biotechnology 14:745(1996)). Other super-binary vectors may be constructed by the methodsset forth in the above references. Super-binary vector pTOK162 iscapable of replication in both E. coli and in A. tumefaciens.Additionally, the vector contains the virB, virC and virG genes from thevirulence region of pTiBo542. The plasmid also contains an antibioticresistance gene, a selectable marker gene, and the nucleic acid ofinterest to be transformed into the plant. The nucleic acid to beinserted into the plant genome is typically located between the twoborder sequences of the T region. Super-binary vectors of the inventioncan be constructed having the features described above for pTOK162.

The T-region of the super-binary vectors and other vectors for use inthe invention are constructed to have restriction sites for theinsertion of the genes to be delivered. Alternatively, the DNA to betransformed can be inserted in the T-DNA region of the vector byutilizing in vivo homologous recombination. See, Herrera-Esterella etal., EMBO J. 2:987 (1983); Horch et al., Science 223:496 (1984). Suchhomologous recombination relies on the fact that the super-binary vectorhas a region homologous with a region of bpR322 or other similarplasmids. Thus, when the two plasmids are brought together, a desiredgene is inserted into the super-binary vector by genetic recombinationvia the homologous regions.

In plants stably transformed by Agrobacteria-mediated transformation,the nucleotide sequence of interest is incorporated into the plantnuclear genome, typically flanked by at least one T-DNA border sequenceand generally two T-DNA border sequences.

Plant cells may be transformed with Agrobacteria by any means known inthe art, e.g., by co-cultivation with cultured isolated protoplasts, ortransformation of intact cells or tissues. The first uses an establishedculture system that allows for culturing protoplasts and subsequentplant regeneration from cultured protoplasts. Identification oftransformed cells or plants is generally accomplished by including aselectable marker in the transforming vector, or by obtaining evidenceof successful bacterial infection.

Protoplasts, which have been transformed by any method known in the art,can also be regenerated to produce intact plants using known techniques.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II,1986). Essentially all plant species can be regenerated from culturedcells or tissues, including but not limited to, all major species ofsugar-cane, sugar beet, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently root.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. It is also advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

The regenerated plants are transferred to standard soil conditions andcultivated in a conventional manner. The plants are grown and harvestedusing conventional procedures.

Alternatively, transgenic plants may be produced using the floral dipmethod (See, e.g., Clough et al., Plant J. 16:735-743 (1998), whichavoids the need for plant tissue culture or regeneration. In onerepresentative protocol, plants are grown in soil until the primaryinflorescence is• about 10 cm tall. The primary inflorescence is cut toinduce the emergence of multiple secondary inflorescences. Theinflorescences of these plants are typically dipped in a suspension ofAgrobacterium containing the vector of interest, a simple sugar (e.g.,sucrose) and surfactant. After the dipping process, the plants are grownto maturity and the seeds are harvested. Transgenic seeds from thesetreated plants can be selected by germination under selective pressure(e.g., using the chemical bialaphos). Transgenic plants containing theselectable marker survive treatment and can be transplanted toindividual pots for subsequent analysis. See, Bechtold, N et ai, MethodsMol Biol. 82:259-266 (1998); Chung et al., Transgenic Res 9:471-476(2000); Clough et al., A. Plant J. 16:735-743 (1998); Mysore et al.,Plant J. 21: 9-16 (2000); Tague et al., Transgenic Res. 10:259-267(2001); Wang et al., Plant Cell Rep 22:274-281 (2003); Ye et al., PlantJ., 19:249-257 (1999).

The particular conditions for transformation, selection and regenerationcan be optimized by those of skill in the art. Factors that affect theefficiency of transformation include the species of plant, the targettissue or cell, composition of the culture media, selectable markergenes, kinds of vectors, and light/dark conditions. Therefore, these andother factors may be varied to determine what is an optimaltransformation protocol for any particular plant species. It isrecognized that not every species will react in the same manner to thetransformation conditions and may require a slightly differentmodification of the protocols disclosed herein. However, by alteringeach of the variables, an optimum protocol can be derived for any plantspecies.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Experimental Procedures Constructs

35SCaMV:HaHB11: A HaHB11 EST was obtained from Tucson University,Arizona (www2.genome.arizona.edu; NCBI Accession No. DY923855; SEQ IDNO:1) and amplified with specific oligonucleotides in order to clone itin the pBI121 vector, under the control of the 358 CaMV promoter.

PrH11:GUS: The promoter region of HaHB11 was isolated from a BAC genomiclibrary using a specific probe. The approximately 125 Kbp BAC insert wasrestricted with several enzymes, electrophoresed and hybridized in aSouthern blot with the same probe. A positive 2000 bp fragment wassubcloned in the pUC119 vector and sequenced. The insert presented 525bp corresponding to the HaHB11 coding sequence plus a 422 bp segmentupstream of the +1 position corresponding to a partial fragment of thepromoter. This fragment was amplified by PCR with specificoligonucleotides and cloned into the pBI101.3 vector. In this way, theHaHB11 promoter directs the expression of the GUS reporter gene. Thisconstruct containing the short 422 bp promoter portion was named PrH11:GUS.

HaHB11 cDNA sequence  (SEQ ID NO: 1)gatgatatatctagtagctagggaggttgaataatagcacacgccactagaggccattggctcttagaattatttatttatttatatatctcgatcaactccc aattggtagttgagaaaatggcagaaaacagtagtagtagtatagagagaaagaagagcaagaagcataacaataggaggttcagcgatgaacaa ataaaatcactggagtcggtgttcaagagggagaacaagctggaaccaaggaagaaggtggagatggctagagagctgggactgcacccgcgccaggtggctatatggtttcaaaacagaagggctcgctggaagtccaaacaagtggagcaagactactacaatctcaaggccgattacgacaccttagct caccgcttcgagtccttaaagaaggagaaacatgccttgctccaccaggtaagtcagctaaaagaactggacagtgggtctgaaaacggaggagagttgaagaatggtaactcaagcagcggaccattagaatacatgcagggtgataaattagtctcagaagaagaagaagaagaaaggcatgaaaaccttg acatggctagtctttttgatcagtcatgttcaaactggtgggacatttggtcatcaaactcatgatcgatattatatatatagcgtagag aattatatatgtata tcttatggggtttgaattgaagtagctagctagctaggatactagttagatatataggaggagctaattaaggatgtaacggcaaagtggtgagcatgtg gatgggcttgctgtttgtttgcactatgcaagatatgtgtgcaaactactactactactagtgtgtcttcacgttcaactcaaatctcatgtgattgcaaactc gatccatcttatttttcgttttcttaatgctatctaacttttgatccaccct

cDNA sequence corresponding to the HaHB11 EST. The start codon (atg) isdouble-underlined and in bold. Underlined are the sequences of theoligonucleotides used to amplify the coding region in order to clone itin the BamHI/SacI sites of the pBI 121 vector.

HaHB11 coding sequence  (SEQ ID NO: 2)atggcagaaaacagtagtagtagtatagagagaaagaagagcaagaagcataacaataggaggttcagcgatgaacaaataaaatcactggagtc ggtgttcaagagggagaacaagctggaaccaaggaagaaggtggagatggctagagagctgggactgcacccgcgccaggtggctatatggtttc aaaacagaagggctcgctggaagtccaaacaagtggagcaagactactacaatctcaaggccgattacgacaccttagctcaccgcttcgagtcctt aaagaaggagaaacatgccttgctccaccaggtaagtcagctaaaagaactggacagtgggtctgaaaacggaggagagttgaagaatggtaactc aagcageggaccattagaatacatgcagggtgataaattagtctcagaagaagaagaagaagaaaggcatgaaaaccttgacatggctagtctttttgatcagtcatgttcaaactggtgggacatttggtcatcaaactcatga The start codon (atg) is double-underlined and in bold.

HaHB11 protein sequence (SEQ ID NO: 3)MAENSSSSIERKKSKKHNNRRFSDEQIKSLESVFKRENKLEPRKKVEMAR ELGLHPRQVAIWF QNRRARWKSKQVEQDYYNLKADYDTLAHRFESLKKEKHALLHQVSQLKEL DSGNGGELK NGNSSSGPLEYMQGDKLVSEEEEEERHENLDMASLFDQSCSNWWDIWSS NSGenomic fragment containing partial HaHB11 promoter sequence (SEQ ID NO: 4) aaaatagaaatattcccatcgatcataaacaattaatatgagtatacataatgaaaactaaccttcaacaccgttgttaattcattttatacgccattccaagtatgcctaggcggtagagattgttgtctttgaaggagaagatcgattaggattcaaaatcctagcatggggtagattagcatgaatatggtataactatgggtaggattatgtaacattcgccgttgcaaaaaaaaaaaaaaaatcatttttatcgtcggtgcacgttttaaggttattaattaataacttgtaactaattgtaagcatcaacacatgttatgtcgtaccagatttttgtattattaattattagtctgctcatgtatatttaataattaataataatcggttaggcatattgtcttccaagtgatatgaataaaatagttgtggaaataagaaaaaggaaatatgattaatgataatatctagtagcta gggaggttgaataatag cacacgccactagaggccattggctcttagaattatttatttatttatatatctcgatcaactcccaattggtagttgagaaaATGGCAGAAAACAGTAGTAGTAGTATAGAGAGAAAGAAGAGCAAGAAGCATAACAATAGCAGCAGTAGGAGGTTCAGCGATGAACAAATAAAATCACTAGAGTCGGTGTTCAAGAGGGAGAACAAGCTGGAACCAAGGAAGAAGGTGGAGATGGCTAGAGAGCTGGGACTGCACCCGCGCCAGGTGGCTATATGGTTTCAAAACAGAAGGGCTCGCTGAAGTCCAAACAGGGGAGCAGACTCTCATTCATGCTTT 

The promoter sequence is in lower case letters. In bold, the 5′ noncoding region. Underlined, the oligonucleotide used to clone thepromoter in the pBI101.3 vector. In capital letters, the cDNA.

Partial HaHB11 promoter sequence  (SEQ ID NO: 5)aaaatagaaatattcccatcgatcataaacaattaatatgagtatacataatgaaaactaaccttcaacaccgttgttaattcattttatacgccattccaagtatgcctaggcggtagagattgttgtctttgaaggagaagatcgattaggattcaaatcctagcatggggtagattagcataatatggtataactatgggtaggattaatgtaacattcggccgttgcaaaaaaaaaaaaaaaatcatttttatcgtcggtgcacgttttaaggttattaattaataacttgtaactaattgtaagcatcaacacatgttatgtcgtaccagatttttgtattattaattattagtctgctcatgtatatttaataattaataataatcggttaggcatattgtcttccaagt Partial HaHB11 promoter sequence and 5′ UTR (SEQ ID NO: 6) aaaatagaaatattcccatcgatcataaacaattaatatgagtatacataatgaaaactaaccttcaacaccgttgttaattcattttatacgccattccaagtatgcctaggcggtagagattgttgtctttgaaggagaagatcgattaggattcaaatcctagcatggggtagattagcatgaatatggtataactatgggtaggattaatgtaacattcgccgttgcaaaaaaaaaaaaaaaatcatttttatcgtcggtgcacgttttaaggttattaattaataacttgtaactaattgtaagcatcaacacatgttatgtcgtaccagatttttgtattattaattattagtctgctcatgtatatttaataattaataataatcggttaggcatattgtcttccaagtgatatgaataaaattagttgtggaaataagaaaaaggaaatatgattaatgataatatctagtagctagggaggttgaataatagcacacgccactagaggccattggctcttagaattatttatttatttatatatctcgatcaactcccaattggtagttgagaaa

Plant Material and Growth Conditions

Arabidopsis thaliana Heyhn. ecotype Columbia (Col-0) was purchased fromLehle Seeds (Tucson, Ariz.). Plants were grown directly on soil in agrowth chamber at 22-24° C. under long-day photoperiods (16 h ofillumination with a mixture of cool-white and Grolux fluorescent lamps)at an intensity of approximately 150 uEm⁻² s⁻¹ in 8 cm diameter×7 cmheight pots during the periods indicated in the figures. Helianthusannuus L (sunflower CF33, from Advanta seeds or HA89 public genotype)seeds were grown on Petri dishes or in soil pots in a culture chamber at28° C. for variable periods of time depending on the purpose of theexperiment as detailed in the figure legends.

Drought Assays

Depending on the experiment and as stated in the figure legends,sunflower seedlings placed on a filter paper or R2 plants grown on soilpots were stressed by cessation of watering during a ten day period.Every two days after this treatment, 1 cm diameter leaf disks werefrozen in liquid nitrogen for further RNA analysis. Twenty five-day-oldArabidopsis plants were subjected to drought stress by cessation ofwatering for 16-18 days. After this treatment, the plants werere-watered until saturation. Survivors were counted two days afterrecovery at normal growth conditions. For water loss evaluation, fiveleaves from four different plants from each genotype were removed at thetimes indicated in the figure and weighed (W1). After that, the sameleaves were incubated in deionized water for 3 h and weighed again (W2).Water loss was estimated as the difference between W1 and W2.

In the case of drought assays maintaining the same water volume, soilpots were weighed every two-three days and water was added as requiredto maintain the same amount of water in each soil pot. The weightdifferences were registered in order to calculate the total water volumeadded to each pot.

Salinity Stress Assays

Salinity stress was applied on sunflower R2 plants grown on soil pots byirrigating them with NaCl 50 mM, NaCl 150 mM (one week later) andfinally, NaCl 200 mM (one more week). After each salt solution addition,1 cm diameter leaf disks were collected and frozen in liquid nitrogenfor further RNA analysis.

Arabidopsis 21-day-old plants grown in normal conditions were saltstressed by adding 1.5 liter of 50 mM NaCl to the whole tray. One weekafter the plants were watered with an additional liter of 150 mM NaCland one additional week after, another liter of 200 mM NaCl was added.In this way, plants in the reproductive stage were salt stressed.

Hormone Treatments

Sunflower seedlings (7-day-old) growing on wet paper were placed inliquid media containing the different hormones (in the concentrationsindicated in the figure legends) and incubated for two hours. The plantswere then frozen in liquid nitrogen; total RNA was extracted from eachsample and analyzed by real time RT-PCR as described below.

A similar procedure was followed with 21-day-old plants grown onperlite/vermiculite.

Arabidopsis thaliana Transformation

Transformed Agrobacterium tumefaciens strain LBA4404 was used to obtaintransgenic Arabidopsis plants by the floral dip procedure (Clough andBent, Plant J. 16:735-743 (1998)). Transformed plants were selected onthe basis of kanamycin resistance and positive PCR which was carried outon genomic DNA with specific oligonucleotides. To assess HaHB11expression, real-time RT-PCR were performed on T2 transformants, asdescribed below. Five positive independent lines for each construct(arising from at least two different transformation experiments) wereused to select homozygous T3 and T4 in order to analyze phenotypes andthe expression levels of HaHB11. Plants transformed with pBI101.3 wereused as negative controls (referred to as WT in the figures). For theother constructs, selection was similarly carried out and three-fiveindependent lines chosen for the analysis.

Transient Transformation of Sunflower Leaves

Transient transformation of sunflower leaf discs was carried out asdescribed Manavella and Chan (2009). Sunflower leaves were infiltratedwith 5 ml of Agrobacterium tumefaciens strain LBA4404 and thentransformed with 35S:HaHB11 or 35S:GUS, used as control. Afterinfiltration, plants were placed in the growth chamber for an additional48 hours; 1 cm diameter disks (50 mg each) were excised from theinfiltrated leaves and RNA was then extracted with Trizol (see below).For each gene transcript measurement, two disks coming from differentplants were analyzed and the experiment was repeated at least twice. Inorder to test the efficiency of infiltration in these experiments, GUSreporter gene expression was measured by histochemical GUS staining.

Histochemical GUS Staining

In situ assays of GUS activity were performed as described by Jeffersonet al., EMBO J. 6:3901-3907 (1987). Whole plants were immersed in a 1 mM5-bromo-4-chloro-3-indolyl-b-glucuronic acid solution in 100 mM sodiumphosphate pH 7.0 and 0.1% Triton X-100 and, after applying vacuum for 5min, they were incubated at 37° C. overnight. Chlorophyll was clearedfrom green plant tissues by immersing them in 70% ethanol.

RNA Isolation and Expression Analyses by Real Time RT-PCR

RNA for real-time RT-PCR was prepared with TRIZOL® reagent (Invitrogen™)according to the manufacturer's instructions. RNA (2 ug) was used forthe RT reactions using M-MLV reverse transcriptase (Promega).Quantitative PCRs were carried out using a MJ-Chromo4 apparatus in a 20ul final volume containing 1 ul SyBr green (10×), 8 pmol of each primer,2 mM MgCl₂, 10 ul of a 1/25 dilution of the RT reaction and 0.12 ulPlatinum Taq (Invitrogen Inc.). Fluorescence was measured at 78-80° C.during 40 cycles. Sunflower RNA was also prepared with the TRIZOL®(Invitrogen Inc.) technique, but in this case the dilution of the RTreaction was 1/50.

Specific oligonucleotides to amply each gene were designed (data notshown).

Chlorophyll Measurements

Extracts from 100 mg of leaves were prepared after freezing with liquidnitrogen. To each sample, 1.5 ml of 80% acetone were added, and thetubes placed in the darkness during 30 min. During this incubation thesample solids were decanted, and the absorbance at 645 and 663 wasmeasured in the supernatants with a spectrophotometer. Chlorophyllconcentration was quantified according to Whatley et al., Nature10:705-708 (1963).

Stomatal Closure

Stomatal closure was quantified on drought stressed plants grown on MSplates, MS supplemented with 10 uM ABA or soil pots. Controls wereperformed with plants grown at normal conditions.

An adhesive was glued on the abaxial side of leaves; the adhesive wasthen carefully detached and glued over a microscope slide in order totake photographs and quantify the open and closed stomata.

Water Loss Treatment

During the drought or desubmergence assay, one leaf from five to sixdifferent plants was removed at the time points indicated in the figurelegend. Subsequently, leaves were weighed (W1), incubated indemineralized water for 3 h, and weighed again (W2). Water loss rate wascalculated as (W2−W1)/W2.

Flooding Stress

To perform submergence assays, 25-day-old plants were completelysubmerged with 3 cm of water over the aerial part of the plant during6-8 days. In waterlogging assays, only the roots were submerged byfilling the pots with water to a height of 7 cm, the pots being 8 cmheight.

Photographs were taken and survival rates were calculated after 6 daysof recovery.

Example 2 HaHB11 Expression Pattern

The sunflower gene HaHB11 encodes a 175 amino acid protein belonging tothe HD-Zip I subfamily of transcription factors (see schematic in FIG.1A and FIG. B; SEQ ID NO:3). In order to characterize HaHB11 geneexpression, total RNA was isolated from 7- and 21-day-old sunflowerplants and analyzed by qRT-PCR as described in Example 1. FIG. 3 showsthat in 7-day-old seedlings, HaHB11 is mainly expressed in hypocotylsand cotyledons as compared with roots and meristems, while 21-day-oldplants (FIG. 4) showed high expression in petioles as compared withother tissues/organs.

We investigated whether abiotic stress factors like drought, salinityand H₂O₂ (oxidative stress generator), and some phytohormones related tostress responses regulate the expression of HaHB11. FIG. 6 and FIG. 7show that in sunflower day-old seedlings, this gene is up-regulated bydrought and 6-benzylaminopurine (BAP) and to a lesser extent by abscisicacid (ABA) and gibberellic acid (GA), while in 21-day-old leaves,mannitol (osmotic factor) and ABA seemed to be the major regulators.Other phytohormones tested (indoleacetic acid [IAA]; ethylene(1-amnocyclopropane-1-carboxylic acid [ACC]), salicylic acid [SA],jasmonate [JA] and GA) exhibited either repression, slight induction orno effect on HaHB11 expression.

Altogether, the results indicated that HaHB11 expression is induced byabiotic stress factors, especially by drought and by the hormone relatedto drought, ABA.

It was observed during these initial studies that drought provoked thehighest induction in HaHB11 expression. Because drought stress isusually related to salt stress, we carried out further investigations inorder to evaluate the effect of drought and salt stresses on HaHB11 geneexpression.

As can be appreciated from FIG. 7, HaHB11 increased its expression untilthe 101 day after initiation of drought treatment. At this time, theplants were very damaged in appearance, and it was not feasible tocontinue with RNA analysis. As a control, HaHB4, a well characterizeddrought responsive gene, was used as a drought marker (data not shown).The expression kinetics of both genes during drought stress were verysimilar. When a high NaCl concentration (as a salt stress generator) wasapplied, HaHB11 increased until three days after starting the treatment(see Example 1), and thereafter declined.

Example 3 Obtaining and Characterization of Arabidopsis TransgenicPlants Ectopically Expressing HaHB11

Aiming to analyze HaHB11 function, transgenic (TG) Arabidopsis plantsexpressing this sunflower gene were obtained. The coding region ofHaHB11 was fused to the 35S CaMV promoter and this construct was used totransform Arabidopsis thaliana plants. Several homozygous lines wererecovered and among them, three named thereafter 35S:HaHB11-A, —B, and—C representing high, medium and low expression, respectively, wereselected for further analysis (FIG. 8).

The transformed plants showed different morphological features whencompared with their WT counterparts. Their leaves were more rounded andsmaller, the petioles were shorter, and the number of leaves was higherin the transgenic genotypes during the transition from the vegetative tothe reproductive phase (FIG. 9).

Besides the different morphology, transgenic plants exhibited aconsiderable delay in their life cycle as assessed by stem elongationand flowering time (FIG. 10). At the end of the cycle, transgenic plantswere taller than their WT counterparts.

The inflorescence morphology of the transgenic genotypes also presentedsignificant differences (FIG. 11). Transgenic flowers were more compactthan in WT plants.

Furthermore, transgenic plants grown on Murashige and Skoog (MS) platesshowed more rounded cotyledons, shorter hypocotyls and larger roots.These phenotypic characteristics were very similar to those observed inWT plants grown in 100 mM mannitol (osmotic stress). Notably, transgenicplants did not alter their phenotype when subjected to osmotic stress,suggesting that they were constitutively prepared for such stress (FIG.12).

Hypocotyl and root length were quantified in order to furthercharacterize these developmental features (FIG. 13).

Example 4 Transgenic Plants Bearing the Construct 35S:HaHB11 were MoreTolerant to Several Abiotic Stress Factors

Having determined that HaHB11 expression was up-regulated by drought,mannitol and ABA, we investigated how transgenic plants (35S:HaHB11)responded to drought at different developmental stages.

Arabidopsis 25-day-old plants were subjected to a severe droughttreatment by stopping the water supply during 15 consecutive days.Transgenic plants showed an enhanced tolerance to this treatment and thepercentage of survivors after watering was significantly higher than forWT plants (FIG. 14). Subjected to this severe treatment, 67-95% of thetransgenic plants survived (depending on the transgene expression level)as compared with 12% of WT plants.

In order to elucidate the physiological mechanisms by which HaHB11conferred drought tolerance, we investigated if transgenic plantsconsumed less water to live or lost less water due to a lowertranspiration rate. To this end, water loss in leaves was quantified inthe transgenic and WT genotypes during a drought stress assay (FIG. 15).Water loss was defined as the weight ratio between dehydrated leaves atdifferent times during the drought stress and the same leaves afterrewatering.

As demonstrated in FIG. 15, 35S:HaHB11 plants lost less water than WTduring the stress treatment, suggesting that their ability to survivecould be due to their capacity to retain water under drought stress.

The best known physiological mechanism by which plants are able toretain water is by stomata closure. We therefore decided to observeplant stomatal closure under water stress.

When plants were subjected to drought stress, transgenic plants closedtheir stomata faster than WT plants (FIG. 16) explaining the resultsshown in FIG. 15.

To further analyze this response, stomata closure/opening was analyzedin plants grown in soil and subjected to drought.

As can be appreciated from FIG. 17, 35S:HaHB11 plants closed 50% oftheir stomata the first day of the drought treatment while WT plantsachieved the same percentage on the 8th day (FIG. 17), indicating thattransgenic plants were able to more quickly sense the stress and,consequently, close their stomata.

Because ABA is intimately related to stomatal closure we decided toevaluate if the more rapid stomata closure observed in transgenic plantswas related to this hormone action. Plants were grown on MS and treatedwith ABA for 1 hour.

ABA treatment provoked similar results as drought; 65-86% of transgenicstomata were closed after one hour while only 22-28% WT stomata wereclosed after the same period (FIG. 18).

When water stress treatments were performed with 35S:HaHB11 plants, weobserved a remarkable difference in the humidity state of the potsbearing these plants as compared with the pots bearing WT plants; theywere always more wet. This interesting observation suggested that,besides the lower water loss, an additional mechanism implying a lowerwater uptake under stress conditions could be playing a role in droughttolerance triggered by HaHB11. Aiming to test this hypothesis, waterconsumption was analyzed in transgenic and WT plants during a droughttreatment.

For this purpose, 25-day-old plants were subjected to drought bycessation of watering. Every day thereafter, the pot was weighed. FIG.19 shows that the pots bearing the transgenic lines with high and mediumexpression HaHB11 levels retained more water (were heavier) than thosebearing WT or transgenic plants with low level HaHB11 expression. Theseresults suggested that transgenics were able to tolerate drought notonly because they closed their stomata faster than WT plants but becausethey had the ability to survive with a lower water supply.

To further investigate the drought tolerance mechanism in HaHB11 plantsand to corroborate if such tolerance resulted from combined orcooperative responses including stomata closure and optimized efficiencyin water use, a drought assay in which the same amount of water wasmaintained in the pots was carried out. FIG. 20 shows that WT plantsneeded more water (twice) than 35S:HaHB11 plants during the assay tomaintain the same pot weight. Although the stress applied was lesssevere, when survivors were counted at the end, there were fewersurviving WT than transgenic plants (FIG. 20).

Altogether, the results indicated a dual tolerance mechanism triggeredin 35S:HaHB11 plants. Under severe drought conditions, transgenic plantsexhibited more efficient water usage due to both lower transpiration andlower consumption rates.

Example 5 Transgenic 35S: HaHB11 Arabidopsis Exhibited Increased YieldUnder Control Conditions

Stomata closure is a natural mechanism of drought tolerance triggered byplants, and several genes are involved. Normally, stomata closure is afirst response when plants sense the drought and results in a loss inproductivity due to the decrease in the photosynthetic rate. Asignificant problem for farmers is that climate and humidity cannot bepredicted with certainty and, as a consequence, drought tolerant plantsthat produce less in watered seasons are not convenient and hence, suchplants are less desirable. Since in most cases in the field, droughtstress is mild rather than severe, we evaluated how transgenic plantsbehave in non-stressed conditions. Yield of 35S:HaHB11 transgenicArabidopsis and wild plants was quantified in standard growth conditionsand the results shown in FIGS. 21A, 21B, and 21C. Under these standardgrowth conditions, transgenic plants had a larger rosette and, as aconsequence, a higher yield of seed.

Example 6 A Complex Molecular Mechanism Takes Place in HaHB11 Plants toTrigger Drought Tolerance

ABA biosynthesis and signaling pathways are intimately related to mostknown mechanisms of abiotic stress tolerance. Aiming to evaluate thephenotype of HaHB11 plants in more detail, the expression levels ofgenes participating in ABA biosynthesis and signaling and those involvedin stress tolerance were quantified in HaHB11 overexpressors treated oruntreated with ABA. RNA from transgenic and WT genotypes was isolatedfrom control or ABA treated plants, and 11 selected gene transcriptswere quantified. FIG. 22 shows the results obtained from threeindependent assays. Transcript levels of five genes involved in ABAbiosynthesis and signaling (ABA1, ABA2, ABI1, ABI2, ABI5) wereconsistently and significantly lower in ABA-treated 35S:HaHB11 plantsthan in WT (FIG. 22).

COR (COR15A and COR47) and RD29A genes have been reported to play keyroles in the abiotic stress response as protectors. Their transcriptlevels were significantly reduced in 35S:HaHB11 plants compared with WT(FIG. 23).

On the other hand, the expression levels of RD29B, EM6 and RAB18 werehigher in 35S:HaHB11 than in WT plants (FIG. 23).

These results indicate that HaHB11 triggers a complex pathway in whichgenes involved in ABA biosynthesis and ABA-independent signalingpathways are down-regulated after drought, salt or cold induction, whilegenes involved in ABA-dependent signaling pathways are up-regulated. Theup-regulation of HaHB11 by ABA and the down-regulation of ABAbiosynthesis genes is suggestive of a negative feedback that regulatesABA concentration after stress.

Example 7 Transgenic Plants Bearing the Construct 35S:HaHB11 were MoreTolerant to Salt Stress

Since stress caused by drought and high salinity are usually related andtaking into account the up-regulation of HaHB11 by salt, transgenicplants were tested for their ability to tolerate salt stress as comparedwith WT plants. Twenty one-day-old 35S:HaHB11 and WT plants were wateredwith increasing NaCl concentrations up to 400 mM for 21 days. As shownin FIG. 24, transgenic plants looked healthier during the treatment thantheir controls and at the end, exhibited a higher survival rate.

Example 8 Isolation and Partial Characterization of 466 bp Fragment ofthe HaHB11 Promoter

A 466 bp segment upstream from the transcription initiation site andcorresponding to the 5′ non coding sequence and a partial promotersegment of HaHB11 was isolated from a BACs genomic library (ProH11short; SEQ ID NO:5). Within this DNA segment different cis-elements wereidentified using the PLACE database (at world wide web sitedna.affrc.go.jp/PLACE) including a LTRE (Low Temperature ResponsiveElement), a DRE (Dehydration Responsive Element) and an ABRE (ABAResponsive Element) located at 318 and 326 bp from the +1 (position 266of SEQ ID NO:4 (−strand); see, schematic in FIG. 1A).

This DNA fragment was cloned to direct expression of the GUS reportergene as described in Example 1. This construct was used to transformArabidopsis plants, which were then analyzed by histochemistry. FIG. 25and FIG. 26 show GUS expression pattern as directed by the HaHB11 shortpromoter fragment. As it can be observed, GUS is expressed in thehypocotyl of Arabidopsis seedlings and in the roots and meristematicregions of 25-day-old plants.

Example 9 HaHB11 Transgenic Plants were Tolerant to Submergence

In order to further investigate the molecular mechanism by which HaHB11conferred drought tolerance to transgenic plants, an exploratorytranscriptome analysis was performed with RNA obtained from sunflowertransiently transformed leaf disks. This analysis indicated that severalgenes were up-regulated such as ADH (Alcohol dehydrogenase), PDC(Pyruvate Decarboxylase) and SS (Sucrose synthase), which are putativehomologs to Arabidopsis genes reported to be involved in submergencetolerance.

In view of these observations, we decided to evaluate the performance ofHaHB11 as a transgene during a stress caused by submergence. Twentyone-day-old plants were submerged in water during six consecutive days.After that, the plants were placed in a dry tray to recover themselvesand were normally irrigated thereafter. FIG. 27A shows the plants afterrecovery; as it can be appreciated, HaHB11 plants tolerated this severestress better and exhibited a higher survival rate than their controls.Moreover, the chlorophyll content of transgenic plants immediately afterthe treatment was higher than in control plants (FIG. 27B) indicatingthat the transgenic plants were somehow protected from the stress andcould better preserve cell and organelle integrity.

Submerged plants do not only suffer during submergence but also duringdesubmergence by dehydration. Cell death is more likely to occur duringthis after stress period. In order to evaluate transgenic plant behaviorafter submergence treatment, water loss was quantified in bothgenotypes, WT and transgenic. FIG. 27C shows the obtained dataindicating that transgenic plants were able to better tolerate thisafter stress period than their non-transformed counterparts.

Example 10 HaHB11 Transgenic Plants are Tolerant to Waterlogging

Waterlogging, caused by flooding, long periods of rain and poor drainageis a serious constraint with damaging effects (Visser et al., Ann. Bot.91:107-109 (2003)). The imbalance between slow diffusion and rapidconsumption of oxygen in plant roots drastically reduces the oxygensupply (Zou et al., Plant Biol. 10:189-215 (2010)). The rapidly depletedoxygen from the submerged root zone is sensed by the plant, whichadjusts the expression of genes induced by anaerobiosis. Floodinginduces or accelerates plant senescence in tobacco, tomato, sunflower,barley, peas, wheat, maize and soybean (Burrows et al., Plant Physiol.22:1105-1112 (1969); Drew and Sisworo, New Phytol. 79:567-571 (1977);Olymbios et al., J. Am. Soc. Hort. Sci. 52:485-500 (1977); Jackson, Sci.Food Agric. 30:143-152 (1979); Trought et al., Plant Soil 54:77-94(1980); VanToai et al., Crop Sci. 34:1112-1115 (1994)). The most obvioussymptom of flooding injury is leaf chlorosis, which is followed bynecrosis, defoliation, cessation of growth and premature plant death.

To evaluate the damage induced by waterlogging, chlorophyll contentafter waterlogging stress was quantified in the different genotypes.FIG. 28 shows that WT plants exhibited a chlorophyll concentration of440-480 ug/g tissue after the treatment, while 35S:HaHB11 plants showedan increased chlorophyll concentration (495-535 ug/g tissue).

It has been reported that many plants that survive flooding dieimmediately afterwards (Sullivan et al., Crop Sci. 41:1-8 (2001)). Thus,the post-flooding period can be as injurious as the flooding perioditself. After the waterlogging treatment, 35S:HaHB11 plants grew betterthan WT, indicating that HaHB11 conferred tolerance to theafter-waterlogging stress. FIG. 29 illustrates the appearance of thedifferent genotypes after six days of recovery.

Example 11 HaHB11 is a Sunflower Divergent Transcription Factor

HaHB11 encodes a 175 amino acid protein belonging to subfamily I ofHD-Zip transcription factors (Arce et al., BMC Plant Biol. 11:42(2011)). Phylogenetic trees based on the HD-Zip domain family ofproteins from several species suggested this protein belongs in the samegroup as AtHB7 and AtHB12 from Arabidopsis thaliana, HaHB4 fromHelianthus annuus and MtHB1 from Medicago truncatula (Chan et al.,Biochim. Biophys. Acta 1442:1-19 (1998); Ariel et al., Trends Plant Sci.12:419-426 (2007); Ariel et al., Plant Cell 22:2171-2183 (2010)).However, more recent phylogenetic trees indicate that neither HaHB11 norHaHB4 can be resolved into the same group as these putative homologs;both sunflower genes are divergent and were not resolved into any of thesix HD-Zip I groups in which this subfamily has been divided based onstructural features outside of the conserved HD-Zip domain (Arce et al.,BMC Plant Biol. 11:42 (2011)). Thus it was interesting to functionallycharacterize these genes that lack orthologs in model species.

The expression pattern of HaHB11 was analyzed in 7- and 21-day oldsunflower plants and by obtaining transgenic Arabidopsis bearing a 452bp promoter segment directing the reporter gen GUS. The results obtainedwith both methodologies indicated that HaHB11 is expressed in hypocotylsand cotyledons, in seedlings and significantly in 21-day-old roots. Thisexpression was up-regulated by drought, mannitol, NaCl and ABA. Thispattern presented similarities to those of AtHB7 and AtHB12 which werealso up regulated by drought (Olsson et al., Plant Mol. Biol. 55:663-677(2004)), NaCl and ABA (Shin et al., Biochem. Biophys. Res. Commun.323:534-40 (2004); and Henriksson et al., Plant Physiol. 139:509-518(2005)). In particular, the expression in leaves and roots was similarto that of AtHB7, while AtHB12 expression was weak in roots, at leastunder standard growth conditions (Henriksson et al., Plant Physiol.139:509-518 (2005)).

The Medicago truncatula gene, MtHB1, was expressed in the root apicalmeristem as well as in the lateral roots emergence region (Ariel et al.,Plant Cell 22:2171-2183 (2010)) in contrast with HaHB11 which isexpressed in the entire root with the exception of the apical meristem.

The expression of GUS directed by the HaHB4 promoter was evident in theroot central cylinder and in the lateral roots emergence region (Dezaret al., Plant Sci. 169:447-459 (2005)) and the expression of this genewas up-regulated by drought and ABA as was the case for all the genesdiscussed above including HaHB11.

35S:HaHB11 plants demonstrated stomata closure under stress conditionsor in response to ABA treatment. Furthermore, seeds were more sensitiveto this hormone in the germination stage. The plant studies indicated anenhanced synthesis or sensitivity to ABA. However, transcriptquantification from genes involved in these processes (synthesis and ABAperception) suggested a different, more complex scenario.

ABA1, ABA2 and ABA3 transcripts have been reported to be induced bywater and salt stresses, contributing to the enhancement of ABA levelsin plants affected by such stresses (Ding et al., J. Genet. Genom.36:17-29 (2009)). The expression of these genes was lowered in HaHB11plants, suggesting that ABA synthesis in these plants is diminished.ABII and ABI2 were reported as negative regulators of the ABA response,and plants over-expressing these genes presented a reduced sensitivityto the hormone (Raghavendra et al., Cell 15:395-401 (2010)). Since thesetwo genes were repressed in HaHB11 plants, the enhanced sensitivity toABA in these plants could be explained in this way.

ABI5 is a transcription factor belonging to the bZip family and it wasreported as a positive regulator of the ABA response. We expected toobserve AB/5 over-expressed in HaHB11 plants; however, the expression ofthis gene was repressed under standard growth conditions and, althoughit was normally induced by ABA, it remained repressed in the transgenicplants treated with ABA as compared with WT plants.

ABA induces ABI5 accumulation by transcriptionally inducing expressionof this gene and stabilizing the encoded protein (Lopez-Molina et al.,PNAS 98:4782-4787 (2001)). This stabilized protein was able to bind theABRE cis elements present in the regulatory regions of several ABAresponsive genes (Hirayama and Shinozaki, Trends Plant Sci. 12:343-351(2007)); Carles et al., Plant J. 30:373-383 (2002)). Plantsover-expressing ABI5 were described as more tolerant to differentabiotic stresses (Nakashima and Yamaguchi-Shinozaki, Japan AgriculturalResearch Quarterly. 39:221-229 (2005)), making it difficult tounderstand why it was repressed in HaHB11 plants.

COR15A and COR47 are regulated by the signal cascade mediated by CBFgenes (Thomoshow, Plant Physiol. 125:89-93 (2001)), and they do not needABA to be expressed under abiotic stress conditions (Yamaguchi-Shinozakiand Shinozaki, Plant Cell 6:251-264 (1994)). Transcripts of these twogenes were repressed in HaHB11 over-expressing plants grown in normalconditions. When these plants were subjected to freezing transcriptionlevels of each of these genes did not appear to differ compared to thatobserved in WT plants. These results are in accordance with thosedescribed by Ding et al., J. Genet. Genom. 36:17-29 (2009), in whichMYB15 over-expressers tolerated drought better than WT but not freezingtemperatures. MYB15 over-expressers exhibited repression of either CBFor repression of genes downstream in the CBF cascade, like COR15A, COR47and RD29A.

RD29A and RD29B have been described as ABA responsive genes. RD29A isregulated both in an ABA dependent and ABA independent way. In itspromoter region there are cis acting elements responsive to drought(DRE) and also cis acting elements responsive to ABA (ABRE; Narusaka etal., Plant J 34:137-148 (2003)). Considering these observations, itsregulation by abiotic stresses does not require the presence of ABA andcan be explained by the presence of DRE elements (Yamaguchi-Shinozaki;Plant Cell 6:251-264 (1994)). On the other hand, RD29A has beendescribed as exclusive to the ABA signaling pathway. This gene wasrepressed in 35S:HaHB11 plants, and this repression remained when theplants were treated with ABA. In contrast, RD29B was induced in35S:HaHB11 plants grown under normal conditions and this inductionincreased 20-40 fold with ABA treatment. These results are in agreementwith those obtained by measuring COR transcripts and indicate thatHaHB11 is a repressor of the ABA independent pathway and an inducer ofthe ABA dependent one.

EM6 encodes a LEA protein involved in seed maturation and is necessaryfor normal seed development (Manfre et al., Plant Physiol. 140:140-149(2005)). EM6 transcripts are induced in HaHB11 plants grown understandard conditions and increase even more when the plants are treatedwith ABA. ABI5 binds an ABRE located in the EM6 promoter (Carles et al.,Plant J. 30:373-383 (2002)), and AB15 was repressed in HaHB11 plants,suggesting that HaHB11 positively regulates EM6 by an ABI5 independentpathway.

RAB18 is repressed in HaHB11 plants both in normal conditions or whentreated with ABA. Surprisingly, RAB18 transcripts were not modified inABA treated WT plants in our experiments although others authors havepreviously reported that this gene is up-regulated by ABA and drought(Lang et al., Plant Mol. Biol. 20:951-962 (1992)). RAB18 up-regulationwas related with ADH1 and DREB1A induction and with drought tolerance(Kyu Hong et al., Planta 227:539-558 (2008)). Furthermore, the inductionof DREB1A resulted in the up-regulation of RD29A (Liu et al., Plant CelltO:1391-1406 (1998)). On the other hand, MYB44 over-expressors weredescribed as drought tolerant exhibiting a rapid stomata closure understress conditions (salinity or drought). In HaHB11 plants RD29A, RD22and RAB18 were repressed indicating a different pathway triggered inthese plants resulting in enhanced tolerance to abiotic stresses.

CBF genes are well-known as involved in cold response as positiveregulators and these genes are repressed in HaHB11 plants as well asother genes described as CBF downstream regulated genes(Yamaguchi-Shinozaki et al., Ann. Rev. Plant Biol. 57:781-803 (2006)).These genes were also repressed in HaHB11 plants. Altogether, theseresults suggest that HaHB11 may function in separate transcriptionalregulation schemes. When induced by drought and salt stresses, HaHB11functions as a positive regulator to aid ABA dependent up-regulation ofstress responsive genes, leading to enhanced drought and salt tolerance,while when this gene is induced by cold stress it would act as anegative controller of an ABA independent pathway. This complexregulation mechanism is summarized in a putative model presented in FIG.30.

Although not all drought tolerant plants are able to toleratesubmergence, it appears that HaHB11 plants can tolerate this stressbecause they are prepared to survive under drought stress by stomataclosing. Two different mechanisms in order to tolerate flooding havebeen described based on experimental data obtained with rice cultivars.One of them involves two AP2/ERF transcription factors named SNORKEL1(SK1) and SK2 (Hattori et al., Ann. Rev. Plant Biol. 57:781-803 (2009))and is essentially an escape response in which internode elongation ispromoted in order to protrude above the surface of the water. The secondone is a quiescent response in which elongation growth is restrained,economizing carbohydrate reserves in order to enable development of newleaves upon desubmergence. This mechanism is regulated by anotherAP2/ERF transcription factor, named submergence inducible gene (SUB1A;Xu et al., Nature 442(7103):705-708 (2006)). Since HaHB11 plants did notpromote internode elongation under submergence stress, while betterresisting this kind of stress, and capable of developing new healthyleaves after desubmergence, it appears that HaHB11 participates in thequiescent response.

Through the phenotypic, physiological and molecular analyses describedabove, positive correlations were found between HaHB11 over-expression,enhanced ABA sensitivity, reduced water loss rate, repression of genesinvolved in ABA biosynthesis and signaling, and induction of othersencoding stress-protective proteins. These correlated changes caused bythe HaHB11 transgene resulted in an improved tolerance to drought, highsalinity stress, waterlogging and submergence and, importantly, improvedyield. Consequently, HaHB11 can be characterized as a positive regulatorof the abiotic stress response.

Example 12 Experimental Procedures Isolation and MolecularCharacterization of Long Portion of HaHB11 Promoter

A long portion of the HaHB11 promoter was isolated from a genomic BAC(Id. 175N08—from Clemson University world wide web site atgenome.clemson.edu). The Sunflower Library ID is HA_HBa and wasgenerated from the ecotype HA383. Two oligonucleotides were designed toisolate 1363 bp of the HaHB11 promoter region which was cloned in theHindIII/XbaI sites of the pUC19 vector, the pBI 121 vector replacing the35S promoter directing HaHB11 cDNA expression and in the pBI 101 vectordirecting GUS expression.

Cloning of HaHB11 Variants from Two Helianthus Cultivars.

The sequence encoding HaHB11 from BAC 175N08 (HaHB11.1, SEQ. ID NO:2)was determined and compared with that isolated from clone RAFL(HaHB11.2, SEQ. ID NO:9). As depicted in FIG. 2A and FIG. 2C, thesequences of these two Helianthus cultivars have differences that resultin differences in the sequences of their respective encoded HaHB11proteins (see, FIG. 2A and FIG. 2B). The most significant differencesbetween the sequences of these encoded HaHB11 proteins result in theinsertions of three consecutive serines and three consecutive glutamicacids in different regions of the BAC 175N08 coding sequence.

Generation of Transgenic Arabidopsis Plants

Proh11 (Long)::H11.1

The HaHB11 long promoter isolated from BAC 175N08 (“ProH11”; SEQ IDNO:16) was inserted between HindIII and XbaI sites of the pBI121 binaryvector, thereby replacing the constitutive 35S promoter and operablyassociating the ProH11 (long) promoter and HaHB11.1 coding sequence (SEQID NO:2). The resulting ProH11 (long)::H11.1 construct was transformedinto E. coli and once confirmed, introduced into Agrobacteriumtummefaciens. Arabidopsis plants, ecotype Col 0, were transformedfollowing the floral dip method.

ProH11 (long)::GUS

Following a strategy similar to that described above for the ProH11(long)::H11.1 construct, the ProH11 long promoter was inserted in theHindIII/XbaI sites of the pBI 101 vector, thereby replacing theconstitutive 35S promoter, and operably associating the ProH11 longpromoter and GUS coding sequence. 35S::H11.2

The coding region from BAC 175N08 (HaHB11.2 (SEQ ID NO:10) was cloned inthe BamH1/SacI sites of the pBI 121 vector, doing a chimerical constructin order to delete introns. In this way, the expression of the HaHB11.2coding sequence was directed by the 35S constitutive promoter.

Activation Capacity Evaluated in Yeast Simple Hybrid

Both versions of the HaHB11 coding sequence (i.e., HaHB11.1 (SEQ IDNO:2) and HaHB11.2 (SEQ ID NO:10) were separately cloned in the pGBKT7vector fused to the GAL4 DNA binding domain. Transactivation ability, asß-galactosidase activity quantitation, was tested for these constructsfollowing a liquid culture assay usingOrtho-Nitrophenyl-ß-D-Galactopyranoside (ONPG) as substrate.

Soluble Sugars and Starch Quantitation

Leaf material (50-80 mg) was frozen in liquid N₂. Sucrose, glucose andstarch were quantified in the soluble and residual fractions ofethanol-water extracts. Each sample was powdered in liquid N₂ andextracted in 700 μl of buffer containing 62.5% methanol, 26.8%chloroform, 5.4 mM PO₄ pH 7.5 and 0.1 mM EDTA. The extracts were kept onice during 20 minutes and after an addition of 300 μl of water, theywere vigorous mixed at room temperature and centrifuged for 5 min at13000 rpm. Soluble sugars were quantified in the supernatant whilestarch in the pellet. The supernatant was decanted and evaporated at 40°C. using a speed vac. The pellet was resuspended in 100 μl of water And50 μl were used to quantify glucose. The remaining 50 μl were incubatedwith 71 U of invertase (Roche) at 37° C. for 1 hour in order to measureglucose. Sucrose concentration was calculated from glucosedetermination. Glucose was tested using an enzymatic kit as follows:Glucose+O₂+H₂O→Gluconic acid+H₂O₂; H₂O₂+4-AF+4-hidroxibenzoat 4red→quinonimin. Red quinonimin was measured at 505 nm.

For starch extraction, the pellet was dried at 60° C. for an hour. Then,250 μl of 0.1 N NaOH were added and the tubes were incubated 30 min at70° C. The samples were neutralized with 75 μl of 0.1 N acetic acid andthen centrifuged for 5 min at 13000 rpm. For starch cleavage we added a50-4, aliquot of the supernatant to 8 μl of 50 mM sodium-acetateincubation buffer, pH 5.1, containing 28 units of amyloglucosidase(Roche). The samples were incubated at 37° C. for 16 h and thencentrifuged for 5 min at 13000 rpm and glucose quantified in thesupernatant.

Yield after Moderate Drought Stress

Moderate drought-stress treatments were carried out on plants grownunder standard conditions until they reached a stem of 150 mm. Becauseof the development delay detected as this stage, WTs and TGs plants weresubjected to drought stress starting at different times. In each case,watering was stopped until a moderate stress level was reached.Subsequently, the stress was maintained by watering the pots every dayin order to maintain the same weight in all the pots. In this case, thefield capacity is 150 g of water and soil and the permanent wiltingpoint is 45 g. Moderate stress was set at 100 g of water and soil. Theplants were watered up to 100 g until siliques were filled and they wereready to be harvested. Yield was informed as the seed weight for eachplant.

Yield after Waterlogging

Plants were grown under normal conditions until reaching a stem lengthof approximately 200 mm. Because of the development delay, wild typeplants and transgenic plants were subjected to stress starting atdifferent times but at the same developmental stage. Plants were watereduntil the water level reached the soil limit. This stress was maintainedfor 3 days and then the plants were grown in standard conditions untilsiliques were filled and could be harvested. Yield was informed as theseed weight of each plant.

Example 13 Identification of HaHB11 Orthologs/Variants in the AsteraceaeFamily

Representative members of the Asteraceae family were screened forsequences encoding HaHB11 orthologs. FIG. 2C shows a sequence alignmentof the HaHB11 orthologs and/or variants in the Asteraceae familyidentified by this screen. The alignment includes the sequence of HaHB11orthologs in Helianthus annuus (Hann; SEQ ID NO:3 and SEQ ID NO:10),Helianthus tuberosus, (Htub; SEQ ID NO:3 and SEQ ID NO:11, SEQ ID NO:14and SEQ ID NO:15), Helianthus argophyllus (Harg; SEQ ID NO:12) andHelianthus ciliaris (Hcil; SEQ ID NO:13). Table 1 provides an overviewof domains in the identified HaHB11 orthologs/variants

TABLE 1 Structural domains of HaHB11 orthologs/variants in theAsteraceae family N- C- Asteraceae SEQ terminal HD-LZ HD LZ terminalmember ID NO: region region region Region region H. annuus 3 1-14 15-11015-74 75-110 111-175 HaHB11.1 H. annuus 10 1-14 15-113 15-77 78-113114-181 HaHB11.2 H. tuberosus 1 11 1-14 15-113 15-77 78-113 114-180 H.argophyllus 12 1-14 15-110 15-74 75-110 111-179 H. ciliaris 13 1-1415-110 15-74 75-110 111-177 H. tuberosus 2 14 1-14 15-112 15-76 77-112113-183 H. tuberosus 3 15 1-14 15-111 15-75 76-111 112-186

Example 14

The HaHB11 Promoter Region Contains Stress Responsive Cis-ActingElements

A 1364 bp fragment corresponding to the HaHB11 promoter region wasisolated from BAC 175N08 as described above. This fragment of thepromoter region exhibited cis-acting elements found in promoters thatparticipate in abiotic stress responses. A LTRECOREATCOR15, core of lowtemperature responsive element (LTRE) is present at position 1158 of SEQID NO:16 (−strand); an ABRELATERD1, ABRE (ABA-responsive element)-likesequence at position 1165 of SEQ ID NO:16 (−strand); two ABRERATCAL,ABRE-related sequence at positions 864 and 1164 of SEQ ID NO:16(−strand); a DRE2COREZMRAB17, DRE (Dehydration responsive element) corefound in Zea maize RAB17 gene promoter at position 1158 of SEQ ID NO:16and an ANAERO2CONSENSUS, one of the 16 motifs found in silico inpromoters of 13 anaerobic genes involved in the fermentative pathway, atposition 243 bp SEQ ID NO:16 (−strand).

Sequence of HAHB11 proH11 long promoter portion (1363 bp; SEQ ID NO: 16): ccccaacaaaggtaaaagaaattttaataatggccgtgtaagacaaaccctccagtcgttttcgtaaaagagttggggtgtctaaatttcattcatcacctttctttttctttttcattatttatatttatttttattcatcatgtcaattttgtttgacaaattaggcctagctagctaggtgtccaaaaccaaacgcataacattgcccacaaccatcaacttatgttgacaattgtaaataagctgctagctagcttgttcatcaattatcaaaacacttgtctttgttaatccaaaccatatatcttaaagccggtgtataagatgatcgattcggggaaatgaatcttgattatcatgatatctttacaggtggcaggaaagctagctagctagctaaattcagtgtcttcctccttacgattgagattattgaaatttatttattatgtgaagcaaatgtaatgcatgtgtgaaagcgtactattggaggagccctatcccacatcgaacgaataagaccttccggttgtattatagacataagatcttgggttactccctctatcaccaattggttttagagtggaaccccattcaatacgtacatccttcatttttgtcctaattgaatttgtacttatcatagcatactttgacaaaaacatatacctggattaactacaaatgaacatgatcatctcacatatatatattaccttctctagcttttactatatatatatatatatatatatatatatatattctcatttaagagaacaaatttaagtactatatattaattatagttgagcgttaaatccgtcacgagtcattctttaaatccgtagatacaagtaccgcgtggaattggaaactcattggatccttttggataaatagaaatattcccatcgatcataaacaattaatatgagtatacataatgaaaactaaccttcaacaccgttgttaattcattttatacgccattccaagtatgcctaggcggtagagattgttgtctttgaaggagaagatcgattaggattcaaatcctagcatggggtagattagcatgaatatggtataactatgggtaggattaatgtaacattcgccgttgcaaaaaaaaaaaatcatttttatcgtcggtgcacgttttaaggttattaattaataacttgtaactaattgtaagcatcaacacatgttatgtcgtaccagatttttgtattattaattattagtctgctcatgtatatttaataattaataataatcggttaggcatattgtcttccaagtgatatgaataaaattagttgtggaaataagaaaaaggaaatatgatta

The ProH11 Long Promoter Fragment Directs GUS Expression in Roots,Hypocotyls and Petioles

As described in the experimental procedures, the 1364 bp fragmentcorresponding to the HaHB11 promoter region was inserted upstream of theβ-glucuronidase (GUS) reporter gene and the resulting expressioncassette was transformed into Arabidopsis plants. GUS expression in thetransgenic plants was evident in cotyledons (A), hypocotyls (B),petioles (C) and roots (D) in normal growth conditions (FIG. 33,photographs A to J: cotyledons (A), hypocotyls (B), petioles (C) androots (D) in normal growth conditions).

The ProH11 Long Promoter is Inducible by ABA and Wounding

In view of the stress response associated cis acting elements detectedin the HaHB11 promoter, a GUS expression pattern assay was conducted intransgenic Arabidopsis plants subjected to stress (i.e., ABA). Asdepicted in FIG. 33, photographs A to J, the location of the GUSexpression in the transgenic plants bearing the ProH11 long::GUSconstruct did not change after ABA treatment. However, the intensity ofthe signal significantly increased in all the tissues, indicating anup-regulation of this promoter by ABA in the cotyledons (A and F),hypocotyls (B and G), petioles (C and H) and roots (D-J).

ProH11 Long::H11 Transgenic Plants Exhibit Longer Roots

Transgenic Arabidopsis plants bearing the construct ProH11 long::H11were grown in MS-agar plates and roots were observed 10 days aftergermination. As depicted in FIG. 34, transgenic plants exhibitsignificantly longer roots compared with WT plants and transgenic35S::GUS control plants.

Example 15 Constitutive Expression of HAHB11 in Transgenic Plants

3S::H11.1 Transgenic Plants Exhibit Increased Yield after Waterlogging

While tolerance to a lethal stress has value as a biotechnologicalindicator, moderate stress conditions are more widespread inagriculture, and plants were therefore subjected to relatively moderatestress conditions in our study. HaHB11.1 and WT plants grown undernormal conditions were given a regular watering regime or subjected to amoderate stress (waterlogging for 3 days). After that, plants werenormally watered. This moderate stress treatment did not cause plantdeath; plants were able to flower and set seeds, so that yield could bequantified for each individual plant. FIG. 30 illustrates seed weightobtained for HaHB11 and WT plants subjected to a moderate waterloggingstress. Grown under standard conditions, HaHB11 plants exhibitapproximately twice yield than WT plants. After the stress treatment,both genotypes showed a yield decrease but that of WT was significantlylarger than that of transgenic plants.

3S::H11.1 Transgenic Plants Exhibit Increased Starch at the End of theDay

Rosettes from three-week-old transgenic 35S:HaHB11 and WT plants grownin standard conditions were analyzed for starch content. FIG. 32A showsa qualitative assay using a lugol staining technique. It can beappreciated that transgenic plants exhibit more starch than WT ones.FIG. 32B shows a quantitation of the accumulated starch during the day.This quantification was performed following the technique described inStrand et al., 1999 (Experimental procedures). Transgenic plantsaccumulated almost twice starch than WT.

Example 16

Transactivation of HAHB11.1 and HAHB11.2 in S. cerevisiae

Sacharomyces cerevisiae, strain Y187, were transformed with bothconstructs bearing the different clones of HaHB11 (HaHB11.1 andHaHB11.2). The activation assay was performed as described in themethods section. FIG. 35 indicates that HaHB11 acts as an activator, atleast in the yeast system, and that both versions do not differ in thisability under the conditions tested.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

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The contents of U.S. Application No. 61/594,133; filed Feb. 2, 2012, isherein incorporated by reference in its entirety. Additionally, allpublications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

1-49. (canceled)
 50. A transgenic plant stably transformed with anisolated nucleic acid encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 3 operably associated with a promoter, whereinthe promoter is heterologous to the coding sequence.
 51. The transgenicplant of claim 50, wherein the nucleic acid sequence comprises thenucleotide sequence of SEQ ID NO: 2 or a nucleic acid sequence having atleast 75% sequence identity with SEQ ID NO:
 2. 52. The transgenic plantof claim 50, wherein the nucleic acid is optimized for increasedexpression in the transgenic plant.
 53. The transgenic plant of claim50, wherein the nucleic acid is codon optimized.
 54. The transgenicplant of claim 50, wherein the plant is (a) a monocot, (b) a dicot, or(c) a sunflower, wheat, maize, soybean, rice, alfalfa, or Arabidopsis.55. The transgenic plant of claim 50, wherein the heterologous promoteris a constitutive promoter or an inducible promoter.
 56. The transgenicplant of claim 55, wherein the constitutive promoter is an actinpromoter.
 57. A crop comprising a plurality of the plant of claim 50.58. A seed produced from the transgenic plant of claim 50, wherein theseed comprises the isolated nucleic acid stably incorporated in itsgenome.
 59. A processed product produced from a product harvested fromthe plant of claim
 50. 60. A method of increasing yield and/orincreasing tolerance of a plant to abiotic stress, the method comprisingstably transforming a plant cell with an isolated nucleic acid encodinga polypeptide comprising the amino acid sequence of SEQ ID NO: 3operably associated with a promoter, wherein the promoter isheterologous to the coding sequence.
 61. The method of claim 60, whereinthe nucleic acid is optimized for increased expression in the transgenicplant.
 62. The method of claim 60, wherein the nucleic acid is codonoptimized.
 63. The method of claim 60, wherein the method furthercomprises regenerating a stably transformed plant from the stablytransformed cell.
 64. The method of claim 60, wherein the method furthercomprises expressing the nucleotide sequence in the plant.
 65. Themethod of claim 60, wherein the method further comprises (a) growing theplant under abiotic stress conditions and/or (b) growing the plant undernormal cultivation conditions.
 66. The method of claim 60, wherein theabiotic stress comprises drought, salt stress, waterlogging stress,submergence stress, desubmergence stress, or a combination thereof. 67.The method of claim 60, wherein the plant is (a) a monocot, (b) a dicot,or (c) a sunflower, wheat, maize, soybean, rice, alfalfa, orArabidopsis.
 68. A method of increasing yield and/or increasingtolerance of a plant to abiotic stress, the method comprising stablytransforming a plant cell with an isolated nucleic acid encoding anHaHB11 polypeptide operably associated with a promoter, wherein (i) thepromoter is heterologous to the nucleic acid encoding the HaHB11polypeptide; (ii) the HaHB11 polypeptide has at least 70% amino sequenceidentity to SEQ ID NO: 3; and, (iii) the stably transformed plant a. hasa higher yield than a non-transformed counterpart plant; and/or, b. ismore tolerant than a non-transformed counterpart plant to abioticstress, wherein the abiotic stress is selected from drought, saltstress, waterlogging stress, submergence stress, desubmergence stress,or a combination thereof.
 69. The method of claim 68, wherein thenucleic acid encoding the HaHB11 polypeptide is codon optimized.